Reverse Phase Protein Array Profiling Research Articles

Reverse Phase Protein Array Profiling Identifies Recurrent Protein Expression Patterns of DNA Damage-Related Proteins across Acute and Chronic Leukemia: Samples from Adults and the Children's Oncology Group.

DNA damage response (DNADR) recognition and repair (DDR) pathways affect carcinogenesis and therapy responsiveness in cancers, including leukemia. We measured protein expression levels of 16 DNADR and DDR proteins using the Reverse Phase Protein Array methodology in acute myeloid (AML) (n = 1310), T-cell acute lymphoblastic leukemia (T-ALL) (n = 361) and chronic lymphocytic leukemia (CLL) (n = 795) cases. Clustering analysis identified five protein expression clusters; three were unique compared to normal CD34+ cells. Individual protein expression differed by disease for 14/16 proteins, with five highest in CLL and nine in T-ALL, and by age in T-ALL and AML (six and eleven proteins, respectively), but not CLL (n = 0). Most (96%) of the CLL cases clustered in one cluster; the other 4% were characterized by higher frequencies of deletion 13q and 17p, and fared poorly (p < 0.001). T-ALL predominated in C1 and AML in C5, but both occurred in all four acute-dominated clusters. Protein clusters showed similar implications for survival and remission duration in pediatric and adult T-ALL and AML populations, with C5 doing best in all. In summary, DNADR and DDR protein expression was abnormal in leukemia and formed recurrent clusters that were shared across the leukemias with shared prognostic implications across diseases, and individual proteins showed age- and disease-related differences.

Abstract 415: The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2)

Abstract Ulixertinib (BVD-523) is a first-in-class small molecule inhibitor of ERK1/2 currently being investigated in several oncology clinical trials, both as a single agent and in combination with other anti-cancer therapeutics. Clinical acquired resistance has been described by others for numerous efficacious small molecule agents. Relevant to the MAPK pathway, emergence of resistance to BRAF and MEK inhibitors limits their clinical efficacy. In vitro models of ulixertinib resistance were generated in an endeavor to characterize and predict potential clinical mechanisms of resistance and guide rational combination therapies. The BRAF-V600E mutant melanoma cell line, A375, was cultured in progressively increasing concentrations of ulixertinib or other MAPK pathway inhibitors (dabrafenib and/or trametinib). Drug-resistant A375 clones were readily obtained following growth in high concentrations of dabrafenib or trametinib. In contrast, developing resistance to ulixertinib proved challenging. Eventually, ulixertinib resistant clones emerged and demonstrated varying degrees of cross-resistance to other MAPK pathway inhibitors and displayed bell-shaped dose-response curves indicative of drug addiction for optimal proliferation. To elucidate potential mechanisms of resistance, Reverse Phase Protein Array profiling of 306 proteins was performed on parental A375 and ulixertinib-resistant clones following treatment with ulixertinib at varying concentrations and treatment times. Principal component analysis revealed clustering of resistant clones by ulixertinib treatment conditions (concentrations and duration). Differences between parental A375 and ulixertinib resistant clones were revealed, including components of MAPK, HER2, and autophagy markers. This work begins to tease out potential mechanisms of resistance to ulixertinib and guide potential combination partners that could circumvent acquired resistance. Citation Format: Deborah Knoerzer, Anupama Reddy, David Sorrell, Caroline M. Emery. The characterization of in vitro models demonstrating a significant delay of acquired resistance to ulixertinib (ERK1/2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 415.

Abstract P009: A shared nearest neighbors approach for integrated, multi-platform networks and its application to the exploration of multiomics data from early-stage non-small cell lung cancers

Abstract Background: The ImmunogenomiC prOfiling of Non-small cell lung cancer (NSCLC) Project (ICON) represents an ambitious undertaking to comprehensively characterize immuno-genomic diversity in NSCLC across diverse platforms. The depth and breadth of this cohort presented a unique opportunity to develop a specialized method for multi-platform data integration and exploration, which can be broadly applied to forthcoming large-scale patient profiling studies. Such a holistic approach can unlock insights for therapeutic targets, biomarkers, and treatment plans by providing a more complete view of phenomena driving disease pathogenesis and evolution. Purpose: We developed a novel shared nearest neighbors (SNN) approach to create an integrated network of ICON’s multi-platform data and identified collections of closely related measurements within the resulting network tied to noteworthy patient characteristics, including recurrence and oncogenotype. Methods: The ICON dataset is derived from tumor and normal lung tissue samples collected from 150 patients at time of resection as well as blood samples collected then and at intervals during the year following. Tissue samples underwent RNA-sequencing (RNA-seq), whole exome sequencing, T-cell receptor sequencing, multiplex immunofluorescence for immune cells, and reverse phase protein array profiling; flow cytometry for immune cells was performed on tissue and blood samples. From these data, the ICON data network was built using an integrative approach based on the SNN algorithm in which genes were linked on the basis of their shared top correlates in orthogonal datasets. Results: The ICON data network currently includes over 20,000 genes linked by over 500,000 connections derived from correlations between RNA-seq and orthogonal platforms. We captured established associations between cancer-related genes and examined these along with new ones in the network. To do so, we used the InfoMap algorithm to extract more interpretable sub-networks, termed modules, from the ICON data network. Single sample gene set enrichment scores for each module were used in multivariate analysis to highlight modules linked to clinical characteristics of interest. As an example, we found modules significantly tied to disease recurrence. The most notable of these was strongly associated with metabolic pathways, and other modules associated with platelets and ion channels were also identified. The metabolic pathway module is being explored as a prognostic biomarker, underscoring the opportunites enabled by mining the network. Conclusions: Through the framework developed, we identified modules in the ICON data network significantly associated with important patient characteristics like recurrence and oncogenotype. We are validating the gene sets identified as potential biomarkers and are developing an interactive application to facilitate further mining of the network. Taken together, our SNN network-building approach enables the integration and exploration of patient data from diverse platforms. Citation Format: Stephanie T. Schmidt, Neal Akhave, Alexandre Reuben, Tina Cascone, Jianhua Zhang, Jun Li, Junya Fujimoto, Lauren A. Byers, Beatriz Sanchez-Espiridion, Lixia Diao, Jing Wang, Lorenzo Federico, Marie-Andree Forget, Daniel J McGrail, Annikka Weissferdt, Shiaw-Yih Lin, Younghee Lee, Natalie Vokes, Carmen Behrens, Ignacio I. Wistuba, Andrew Futreal, Ara Vaporciyan, Boris Sepesi, John V. Heymach, Chantale Bernatchez, Cara Haymaker, Jianjun Zhang, Christopher A. Bristow, Timothy P. Heffernan, Marcelo V. Negrao, Don L. Gibbons. A shared nearest neighbors approach for integrated, multi-platform networks and its application to the exploration of multiomics data from early-stage non-small cell lung cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P009.

High-throughput proteomic profiling reveals mechanisms of action of AMG925, a dual FLT3-CDK4/6 kinase inhibitor targeting AML and AML stem/progenitor cells.

FLT3 mutations, which are found in a third of patients with acute myeloid leukemia (AML), are associated with poor prognosis. Responses to currently available FLT3 inhibitors in AML patients are typically transient and followed by disease recurrence. Thus, FLT3 inhibitors with new inhibitory mechanisms are needed to improve therapeutic outcomes. AMG925 is a novel, potent, small-molecule dual inhibitor of FLT3 and CDK4/6. In this study. we determined the antileukemic effects and mechanisms of action of AMG925 in AML cell lines and primary samples, in particular AML stem/progenitor cells. AMG925 inhibited cell growth and promoted apoptosis in AML cells with or without FLT3 mutations. Reverse-phase protein array profiling confirmed its on-target effects on FLT3-CDK4/6-regulated pathways and identified unrevealed signaling network alterations in AML blasts and stem/progenitor cells in response to AMG925. Mass cytometry identified pathways that may confer resistance to AMG925 in phenotypically defined AML stem/progenitor cells and demonstrated that combined blockade of FLT3-CDK4/6 and AKT/mTOR signaling facilitated stem cell death. Our findings provide a rationale for the mechanism-based inhibition of FLT3-CDK4/6 and for combinatorial approaches to improve the efficacy of FLT3 inhibition in both FLT3 wild-type and FLT3-mutated AML.

Immuno-genomic landscape of osteosarcoma

Limited clinical activity has been seen in osteosarcoma (OS) patients treated with immune checkpoint inhibitors (ICI). To gain insights into the immunogenic potential of these tumors, we conducted whole genome, RNA, and T-cell receptor sequencing, immunohistochemistry and reverse phase protein array profiling (RPPA) on OS specimens from 48 pediatric and adult patients with primary, relapsed, and metastatic OS. Median immune infiltrate level was lower than in other tumor types where ICI are effective, with concomitant low T-cell receptor clonalities. Neoantigen expression in OS was lacking and significantly associated with high levels of nonsense-mediated decay (NMD). Samples with low immune infiltrate had higher number of deleted genes while those with high immune infiltrate expressed higher levels of adaptive resistance pathways. PARP2 expression levels were significantly negatively associated with the immune infiltrate. Together, these data reveal multiple immunosuppressive features of OS and suggest immunotherapeutic opportunities in OS patients.

Implementing Reverse Phase Protein Array Profiling as a Sensitive Method for the Early Pre-Clinical Detection of Off-Target Toxicities Associated with Sunitinib Malate.

The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40mgkg-1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ off-target toxicities.

Functional Proteomics of Breast Cancer Metabolism Identifies GLUL as Responder during Hypoxic Adaptation.

Hypoxia as well as metabolism are central hallmarks of cancer, and hypoxia-inducible factors (HIFs) and metabolic effectors are crucial elements in oxygen-compromised tumor environments. Knowledge of changes in the expression of metabolic proteins in response to HIF function could provide mechanistic insights into adaptation to hypoxic stress, tumorigenesis, and disease progression. We analyzed time-resolved alterations in metabolism-associated protein levels in response to different oxygen potentials across breast cancer cell lines. Effects on the cellular metabolism of both HIF-dependent and -independent processes were analyzed by reverse-phase protein array profiling and a custom statistical model. We revealed a strong induction of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) as well as reduced glutamate-ammonia ligase (GLUL) protein levels across all cell lines tested as consistent changes upon hypoxia induction. Low GLUL protein levels were correlated with aggressive molecular subtypes in breast cancer patient data sets and also with hypoxic tumor regions in a xenograft mouse tumor model. Moreover, low GLUL expression was associated with poor survival in breast cancer patients and with high HIF-1α-expressing patient subgroups. Our data reveal time-resolved changes in the regulation of metabolic proteins under oxygen-deprived conditions and elucidate GLUL as a strong responder to HIFs and the hypoxic environment.

Abstract 4627: Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC)

Abstract Metabolic reprogramming, rewired signalling cascades, altered microbiota composition and aberrations in the tumor micro-environment have been implicated in CRC progression and response to treatment. In this study, we integrated proteomics, metabolomics, gut microbiota and predictions from network-analysis in tumor and non-cancerous tissue to build a comprehensive atlas to decipher the associations among alterations that occur in the different molecular layers. We performed our molecular fingerprinting in fresh frozen tissue samples from both tumor (centre and/or junction) and matched mucosa (5 and/or 10 cm distance) prospectively collected from a cohort of n=53 stage 0-IV CRC patients. We quantified the protein expression of a panel of 86 cancer-related targets involved in DNA damage/repair mechanisms, cell cycle regulation, growth/angiogenesis signalling, apoptosis and bioenergetics by Reverse Phase Protein Array (RPPA) in n=157 samples (n=48 from tumor and n=109 from mucosa) from n=53 patients. RPPA profiling on both tumor and matched normal tissue was available for 34 out of 53 patients. We performed High Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HR-MAS NMR) to profile metabolites in n=332 samples from n=52 patients (n=161 and n=171 from tumor and normal tissue, respectively). We characterized gut microbiota features from 16S rRNA sequencing in n=54 samples from n=18 patients. We assessed bioenergetic fitness using an ordinary-differential equations based model of core carbon metabolism that we are developing. The model can simulate the network dynamic and response to fuels (such as glucose, pyruvate and lactate) from extracellular milieu and their utilization and conversion via reactions in the cytoplasm and mitochondrial compartments. We used RPPA-based protein expression for HKII, TIGAR, PGYM, LDHA, MCT4, ATP5A as a proxy for enzymatic activities as case-specific inputs to the model. We identified distinct phenotypic differences when comparing tumors with normal tissue at each data type. Tumors showed higher protein levels for CHK1, GSK3B and LDHA; were enriched with fusobacteria; had increased metabolic levels of glycerphosphocholine, isoglutamine, phosphocholine, taurine and lactate; and were predicted by the mathematical model to have a more glycolytic phenotype. Normal mucosa had higher protein levels for GAB1 and elevated isobutyrate and lipids content. We further analysed the NMR spectra and using a genetic algorithm we identified 10 features (ppm levels) that could classify the tissue type with 88% cross-validated accuracy. We presented a unified analysis connecting dysregulations affecting the proteome, metabolome and microbiome. We provided insights into the heterogeneity of CRC which may lead to more specific molecular classifications and ultimately more targeted treatments. Citation Format: Manuela Salvucci, Liam Poynter, Reza Minerzami, Steven Carberry, Robert O'Byrne, Mattia Cremona, Bryan T. Hennessy, Kirill Veselkov, James Kinross, Jochen H. Prehn. Integrated multi-layer analysis reveals novel insights into the molecular landscape of colorectal cancer (CRC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4627.

Novel biomarkers of nasopharyngeal carcinoma metastasis risk identified by reverse phase protein array based tumor profiling with consideration of plasma Epstein-Barr virus DNA load.

In patients with Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC), intertumor heterogeneity causes interpatient heterogeneity in the risk of distant metastasis. We aimed to identify novel biomarkers of metastasis risk using reverse phase protein array (RPPA) profiling of NPC patients at risk for metastasis and considering plasma EBV DNA load. A total of 98 patients with NPC with and without metastasis after treatment, matched with respect to clinical parameters, are enrolled. Total protein expression is measured by RPPA, and protein functions are analyzed by pathway bioinformatics. The RPPA analysis revealed a profile of 70 proteins that are differentially expressed in metastatic and nonmetastatic tumors. Plasma EBV DNA load after treatment correlated with protein expression level better than plasma EBV DNA load before treatment did. The biomarkers of NPC metastasis identified by proteomics regulate signaling pathways involved in cell cycle progression, apoptosis, and epithelial-mesenchymal transition. The authors identified 26 biomarkers associated with 5-year distant failure-free survival in univariate analysis; five biomarkers remained significant in multivariate analysis. A comprehensive RPPA profiling study is warranted to identify novel metastasis-related biomarkers and further examine the activation state of signaling proteins to improve estimation of metastasis risk for patients with EBV-associated NPC.

AMG925, a Dual FLT3-CDK4/6 Inhibitor, Disrupts Survival Signaling and Triggers Apoptosis in AML Progenitor/Stem Cells

FLT3 mutations are detected in one third of patients diagnosed with acute myeloid leukemia (AML) and confer poor outcomes. Selective FLT3 inhibitors have been developed and are being tested at different stages of clinical development. Drug resistance triggered by inhibitor-induced secondary mutations is often observed, and is a major cause of treatment failure. AMG925 (FLX925) is a small molecule dual FLT3-CDK4/6 inhibitor, currently in clinical development. Because AMG925 concomitantly inhibits both FLT3 and CDK4/6, AMG925 treatment rarely causes development of secondary drug-resistant FLT3 mutations (Li C et. al Mol. Cancer Ther. 2015). In this study, we examined the anti-leukemic effect of AMG925 on AML, focusing on AML progenitor/stem cells. Utilizing the high-throughput technology of time-of-flight mass cytometry (CyTOF) and reverse phase protein array (RPPA), we investigated alterations of the FLT3-mediated signaling network trigged by AMG925 at the single cell level. While AMG925 caused cell growth inhibition and apoptosis in both, FLT3-mutated (MOLM13, MV4;11, Ba/F3-ITD, Ba/F3-D835Y and Ba/F3-ITD/D835Y) and -wild type (Ba/F3-wt, OCI-AML3 and U937) cell lines and primary AML blasts, it elicited maximal response in cells carrying ITD, D835Y and ITD/D835Y double mutations. Mechanistically, AMG925 attenuates CDK4 phosphorylation of retinoblastoma protein (Rb), inhibits FLT3 activation and abrogates FLT3-dependent and independent AKT/mTOR, MEK/ERK and STAT5 signaling. AMG925 at 10nM effectively inhibited colony formation of PDX-derived leukemic stem cells originating from 2 AML patients carrying FLT3-ITD mutation (54±8.7% inhibition, n=4). CyTOF analysis of cell surface and intracellular molecules measured by a panel of 36 antibodies revealed that the inhibitory effect of AMG925 on p-AKT, p-ERK and p-STAT5 is operational primarily in subpopulations expressing the stem cell markers CD123, CD117, CD34, CD133 and CD47. Using RPPA, we studied the expression and phosphorylation changes of 273 proteins from the same 2 AML-ITD xenograft samples in response to AMG925 ex-vivo. Heterogeneous expression signatures were observed in all samples isolated from bone marrows (n=1) and spleens (n=4). AMG925 treatment altered multiple signaling networks by inhibiting key molecules in the AKT/mTOR, cyclin D family, MEK/ERK and Src pathways, and triggered a stress-response by up-regulating STAT3 and TSC1 in all samples examined. The combinational consequences of targeting FLT3-CDK4/6 and the proteins that contribute to resistance are under investigation. Conclusions: combined FLT3-CDK4/6 inhibition by AMG925 results in growth inhibition and apoptosis of AML cells with or without FLT3 mutations and selectively inhibits signaling within PDX and phenotypically defined AML stem/progenitor cell populations. CyTOF analysis confirmed on-target effects of AMG925 on FLT3-regulated signaling in AML stem cell subsets. RPPA profiling of AML-ITD xenografts exposed ex-vivo to AMG925 revealed alterations in multiple pro-survival signaling pathways and identified the upregulation of alternative pathways potentially conferring resistance to AMG925. While our results indicate efficacy of combined FLT3-CDK4/6 inhibition it identifies unexpected resistance mechanisms that provide rationale for the additional mechanism-based combinatorial approaches to increase the therapeutic efficacy in AML DisclosuresShah:ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

505: Reverse phase protein array profiling of lobular and triple negative breast cancers within the RATHER consortium

505: Reverse phase protein array profiling of lobular and triple negative breast cancers within the RATHER consortium

Reverse-Phase Protein Array Profiling of Oropharyngeal Cancer and Significance of PIK3CA Mutations in HPV-Associated Head and Neck Cancer

Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers.

Reverse phase protein array profiling reveals distinct proteomic signatures associated with chronic myeloid leukemia progression and with chronic phase in the CD34‐positive compartment

Chronic myeloid leukemia (CML) is a clonal stem cell malignancy whose pathogenesis is driven by constitutive activation of the breakpoint cluster region-v-abl Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL1) kinase. Although BCR-ABL1 activation is present in all patients with CML, patients can present in 3 different phases characterized by an increasingly worse prognosis and diminished responsiveness to tyrosine kinase inhibitors: chronic phase, accelerated phase, or blastic phase. The biologic basis for progression from chronic phase to blastic phase and for regulating the homeostasis of tyrosine kinase inhibitor-resistant CML stem cells is not entirely understood. To shed some light into these aspects of CML biology, the authors used reverse phase protein arrays probed with 112 individual monoclonal antibodies to compare protein expression patterns in 40 samples of leukemia-enriched fractions from patients with CML (25 in chronic phase, 5 in accelerated phase, and 10 in phase). An analysis of variance (significance cutoff, P < .01) unveiled a set of proteins that were overexpressed in blastic phase, including heat-shock protein 90 (hsp90); retinoblastoma (Rb); apoptosis-inducing factor (AIF); serine/threonine-protein phosphatase 2A (PP2A); B-cell leukemia 2 (Bcl-2); X-linked inhibitor of apoptosis protein (Xiap); human homolog of Drosophila Mad (mothers against decapentaplegic) and related Caenorhabditis elegans gene Sma, family member 1 (Smad1); single-stranded DNA binding protein 2 alpha (SSBP2α); poly(adenosine diphosphate-ribose) polymerase (PARP); GRB2-associated binding protein 2 (Gab2); and tripartite motif containing 24 (Trim24). It is noteworthy that several of these proteins also were overexpressed in the CD34-positive compartment, which putatively contains the CML stem cell population. The results from this study indicated that reverse phase protein array analysis can unveil differentially expressed proteins in advanced phase CML that can be exploited therapeutically with targeted approaches.

The development of an efficient procedure for protein extraction from formalin fixed and paraffin embedded tissues and analysis of its application to reverse phase protein arrays

Abstract Purpose: Formalin fixation coupled with paraffin embedding (FFPE) is the standard tissue fixation and storage method adopted by most health institutions in the world. However, protein extraction from FFPE tissues has been challenging due to the extensive molecular cross-linking that occurs upon formalin fixation. The reverse-phase protein array (RPPA) is a novel high-throughput technology which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations. Reproducibility and reliability of the technique in FFPE samples has not been demonstrated extensively so far. The purpose of this study is to develop and optimize an efficient and reproducible procedure for extraction of proteins from FFPE tissues, and evaluate its performance on RPPA. Materials and Methods: Fresh frozen and FFPE material was obtained from breast cancer cell lines, xenografts, renal and breast cancer patient tissues, as well as normal renal cortex tissue. Serial FFPE cell or tissue sections were deparaffinized and processed using six different protein extraction protocols. The yield and protein quality from these protocols were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie blue staining and Western Blots. Protein lysates were then subjected to reverse-phase protein array profiling by using the most efficient protocol. The Pearson correlation coefficient between replicate preparations or between FFPE and matched frozen samples was calculated, and its statistical significance was determined (R software). Unsupervised hierarchical clustering and heatmaps were generated by the R package Class Discovery. Results: The protein extraction protocol that produced the highest yield and best protein quality included a boiling step in 2mM Tris buffer containing 2% sodium dodecyl sulfate at pH 9.0 for 20 minutes, followed by incubation in 60°C for 2 hours. This method was used to prepare lysates for the RPPA assay. The correlation coefficient between replicate preparations across the tested protein markers demonstrated high reproducibility of the technique, with the majority of the markers showing coefficients equal or more than 0.7. The protocol enables efficient extraction of immunoreactive protein from all four experimental settings, with the majority of protein markers (119 of the 203 tested) showing significant correlation between FFPE and frozen preparations (R&amp;gt;0.5). Protein markers whose expression is specifically influenced by particular cell culture conditions and pharmacological interventions also proved that results derived from FFPE specimens are biologically relevant and reliable. Importantly, the RPPA-based global protein expression profile of the FFPE preparations was able to distinguish between normal renal and tumor samples, and categorize the patient samples into distinct groups slightly more successfully than their frozen counterparts. Conclusions: Our results indicate that, with the optimized protein extraction method, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using reverse-phase protein arrays (RPPA).

Gene and protein expression markers of response to combined antiangiogenic and epidermal growth factor targeted therapy in renal cell carcinoma

BackgroundMetastatic renal cell carcinoma (mRCC) patients treated with anti-vascular endothelial growth factor (VEGF) therapies demonstrate promising outcomes but not all patients benefit. Factors that predict response remain to be elucidated. Patients and methodsNephrectomy material from 37 patients with mRCC receiving bevacizumab ± erlotinib was used for protein and gene expression assessment. Protein lysates were subjected to reverse-phase protein array profiling. RNA extracts were used to carry out gene expression microarray-based profiling. Normalized protein and gene expression data were correlated with overall survival (OS) and progression-free survival (PFS) using univariate Cox hazard model and linear regression. Immunoblotting was carried out to validate the results. ResultsHigh protein levels of AMP-activated protein kinase and low levels of cyclin B1 (CCNB1) were associated with longer OS and PFS. Further validation revealed reduced expression and activation of phosphoinositide 3-kinase (PI3K) pathway components and cell cycle factors in patients with prolonged survival after therapy. Gene expression analysis revealed up-regulation of PI3K- and cell cycle-related pathways in patients with shorter PFS. ConclusionsThe OS and PFS of bevacizumab ± erlotinib-treated patients with renal cell carcinoma were associated with changes in expression of protein and gene expression markers related to PI3K pathway and cell cycle signaling.