Systems analysis of the NCI-60 cancer cell lines by alignment of protein pathway activation modules with "-OMIC" data fields and therapeutic response signatures

The NCI-60 cell line set is likely the most molecularly profiled set of human tumor cell lines in the world. However, a critical missing component of previous analyses has been the inability to place the massive amounts of "-omic" data in the context of functional protein signaling networks, which often contain many of the drug targets for new targeted therapeutics. We used reverse-phase protein array (RPPA) analysis to measure the activation/phosphorylation state of 135 proteins, with a total analysis of nearly 200 key protein isoforms involved in cell proliferation, survival, migration, adhesion, etc., in all 60 cell lines. We aggregated the signaling data into biochemical modules of interconnected kinase substrates for 6 key cancer signaling pathways: AKT, mTOR, EGF receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), integrin, and apoptosis signaling. The net activation state of these protein network modules was correlated to available individual protein, phosphoprotein, mutational, metabolomic, miRNA, transcriptional, and drug sensitivity data. Pathway activation mapping identified reproducible and distinct signaling cohorts that transcended organ-type distinctions. Direct correlations with the protein network modules involved largely protein phosphorylation data but we also identified direct correlations of signaling networks with metabolites, miRNA, and DNA data. The integration of protein activation measurements into biochemically interconnected modules provided a novel means to align the functional protein architecture with multiple "-omic" data sets and therapeutic response correlations. This approach may provide a deeper understanding of how cellular biochemistry defines therapeutic response. Such "-omic" portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches. © 2013 American Association for Cancer Research

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10-6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e-15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein. Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative

A step towards personalizing next line therapy for resected pancreatic and related cancer patients: A single institution\u27s experience

Background: There is a lack of precision medicine in pancreatic ductal adenocarcinoma (PDA) and related cancers, and outcomes for patients with this diagnosis remain poor despite decades of research investigating this disease. Therefore, it is necessary to explore novel therapeutic options for these patients who may benefit from personalized therapies. Objective: Molecular profiling of hepatopancreaticobiliary malignancies at our institution, including but not limited to PDA, was initiated to assess the feasibility of incorporating molecular profiling results into patient oncological therapy planning. Methods: All eligible patients from Thomas Jefferson University (TJU) with hepatopancreaticobiliary tumors including PDA, who agreed to molecular testing profiling, were prospectively enrolled in a registry study from December 2014 to September 2017 and their tumor samples were tested to identify molecular markers that can be used to guide therapy options in the future. Next generation sequencing (NGS) and protein expression in tumor samples were tested at CLIA-certified laboratories. Prospective clinicopathologic data were extracted from medical records and compiled in a de-identified fashion. Results: Seventy eight (78) patients were enrolled in the study, which included 65/78 patients with PDA (local and metastatic) and out of that subset, 52/65 patients had surgically resected PDA. Therapy recommendations were generated based on molecular and clinicopathologic data for all enrolled patients. NGS uncovered actionable alterations in 25/52 surgically resected PDAs (48%) which could be used to guide therapy options in the future. High expression of three proteins, TS (p = 0.005), ERCC1 (p = 0.001), and PD-1 (p = 0.04), was associated with reduced recurrence-free survival (RFS), while TP53 mutations were correlated with longer RFS (p = 0.01). Conclusions: The goal of this study was to implement a stepwise strategy to identify and profile resected PDAs at our institution. Consistent with previous studies, approximately half of patients with resected PDA harbor actionable mutations with possible targeted therapeutic implications. Ongoing studies will determine the clinical value of identifying these mutations in patients with resected PDA

Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPK\uce\ub2 in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF- 02341066/Xalkori\uc2\uae, Pfizer Inc), induced both cytotoxicity (IC50= 0.89 \uce\ubcM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics. \uc2\ua9 2013 Robertson et al

Mass spectrometry-based biomarker discovery

Discovery of candidate biomarkers within the entire proteome is one of the most important and challenging goals in proteomic research. Mass spectrometry-based proteomic is a modern and promising technology for semiquantitative and qualitative assessment of proteins, enabling protein sequencing and identification with exquisite accuracy and sensitivity. For mass spectrometry analysis, protein extractions from tissues of interest or body fluids with subsequent protein fractionation represent an important and unavoidable step in the workflow for biomarker discovery. The aim of our chapter is to provide practical lab procedures for sample digestion and protein fractionation for subsequent mass spectrometry analysis

Cancer Proteomics: The State of the Art

Now that the human genome has been determined, the field of proteomics is ramping up to tackle the vast protein networks that both control and are controlled by the information encoded by the genome. The study of proteomics should yield an unparalleled understanding of cancer as well as an invaluable new target for therapeutic intervention and markers for early detection. This rapidly expanding field attempts to track the protein interactions responsible for all cellular processes. By careful analysis of these systems, a detailed understanding of the molecular causes and consequences of cancer should emerge. A brief overview of some of the cutting edge technologies employed by this rapidly expanding field is given, along with specific examples of how these technologies are employed. Soon cellular protein networks will be understood at a level that will permit a totally new paradigm of diagnosis and will allow therapy tailored to individual patients and situations

Nanoparticle technology: Addressing the fundamental roadblocks to protein biomarker discovery

The incorporation of affinity baits into N-isopropylacrylamide-hydrogel- based nanoparticles offers a novel technology that addresses the major analytical challenges of disease biomarker discovery. In solution in complex biologic fluids (e.g. blood or urine), core-shell bait-containing nanoparticles can perform three functions in one step: (a) sieve molecules according to size, (b) sequestrate and concentrate target analytes, and (c) protect analytes from degradation. © 2009 The Royal Society of Chemistry 2009

The amplified peptidome : the new treasure chest of candidate biomarkers

Mass spectrometric analysis of the low-molecular weight (LMW) range of the serum/plasma proteome is revealing the existence of large numbers of previously unknown peptides and protein fragments predicted to be derived from low- abundance proteins. This raises the question of why such low abundance molecules would be retained at detectable levels in the circulation, instead of being rapidly cleared and excreted. Theoretical models of biomarker production and association with serum carrier proteins have been developed to elucidate the mechanisms governing biomarker half-life in the bloodstream. These models predict that the vast majority of LMW biomarkers exist in association with circulating high molecular mass carrier proteins. Moreover, the total serum/ plasma concentration of the biomarker is largely determined by the clearance rate of the carrier protein, not the free-phase biomarker clearance itself. These predictions have been verified experimentally using molecular mass fractionation of human serum before mass spectrometry sequence analysis. These principles have profound implications for biomarker discovery and measurement