Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

Integration of proteomics into systems biology of cancer.

Deciphering the complexity and heterogeneity of cancer, benefits from integration of proteomic level data into systems biology efforts. The opportunities available as a result of advances in proteomic technologies, the successes to date, and the challenges involved in integrating diverse datasets are addressed in this review.

Mining the plasma proteome for disease applications across seven logs of protein abundance.

The current state of proteomics technologies has sufficiently advanced to allow in-depth quantitative analysis of the plasma proteome and development of a related knowledge base. Here we review approaches that have been applied to increase depth of analysis by mass spectrometry given the substantial complexity of plasma and the vast dynamic range of protein abundance. Fractionation strategies resulting in reduced complexity of individual fractions followed by mass spectrometry analysis of digests from individual fractions has allowed well in excess of 1000 proteins to be identified and quantified with high confidence that span more than seven logs of protein abundance. Such depth of analysis has contributed to elucidation of plasma proteome variation in health and of protein changes associated with disease states.

Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P < 0.01). Down-regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P < 0.001). In situ hybridisation shows that Asb-4 mRNA is expressed in brain areas linked to energy homeostasis, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus and posterodorsal medial amygdaloid area. Double in situ hybridisation revealed that Asb-4 mRNA colocalises with key energy homeostatic neurones. In the fed state, Asb-4 mRNA is expressed by 95.6% of pro-opiomelanocortin (POMC) neurones and 46.4% of neuropeptide Y (NPY) neurones. By contrast, in the fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P < 0.001). Moreover, the density of Asb-4 mRNA per fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.

Multi-dimensional liquid phase based separations in proteomics.

This review covers recent developments towards the implementation of multi-dimensional (MuD) liquid phase based systems for proteome investigations. Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as a standard approach in proteomics, its drawbacks including the limited dynamic range and molecular mass range, together with lack of on-line integration with biological mass spectrometery (Bio-MS) have limited its widespread use and applications in proteomics. In the meantime, various liquid-phase based multi-dimensional separation techniques have been explored. Especially, with the emergence of the combination of nanoflow capillary high-performance liquid chromatography (cHPLC) and Bio-MS, attention is again refocused on utilizing multi-dimensional liquid-phase based separation of proteins. Some remarkable applications of on-line analysis of intact proteins and on-column digested proteins, and the emergence of approaches such as multiple HPLC-electrospray ionization tandem MS and capillary array electrophoresis-matrix assisted laser desorption ionization MS, have stimulated thinking towards developing a automated multi-dimensional system (MuDSy) that integrates liquid phase based separation, digestion and identification of proteins in complex biological mixtures.

Identification of novel targets for cancer therapy using expression proteomics.

Although most drugs target proteins, the proteome has remained largely untapped for the discovery of drug targets. The sequencing of the human genome has had a tremendous impact on proteomics and has provided a framework for protein identification. There is currently substantial interest in implementing proteomics platforms for drug target discovery. Although the field is still in the early stages, current proteomic tools include a variety of technologies that could be implemented for large-scale protein expression analysis of cells and tissues, leading to discovery of novel drug targets. Proteomics uniquely allows delineation of global changes in protein expression patterns resulting from transcriptional and post-transcriptional control, post-translational modifications and shifts in proteins between different cellular compartments. Some of the current technologies for proteome profiling and the application of proteomics to the analysis of leukemias by our group are reviewed.

Contributions of proteome profiling to the molecular analysis of cancer.

The proteome is the most functional compartment encoded for in the genome. Technologies for protein separation and quantitation, coupled with mass spectrometry for protein identification, have provided the means for proteome profiling of tumor cell lines and tissues that complement genomic and transcriptomic profiling. The application of established and novel proteomic technologies to the molecular analysis of cancer is reviewed.

DNA methyltransferase 3B mutations linked to the ICF syndrome cause dysregulation of lymphogenesis genes.

ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small decrease in overall genomic 5-methylcytosine levels and much hypomethylation of Chr1 and Chr16 juxtacentromeric heterochromatin. Microarray expression analysis was done on B-cell lymphoblastoid cell lines (LCLs) from ICF patients with diverse DNMT3B mutations and on control LCLs using oligonucleotide arrays for approximately 5600 different genes, 510 of which showed a lymphoid lineage-restricted expression pattern among several different lineages tested. A set of 32 genes had consistent and significant ICF-specific changes in RNA levels. Half of these genes play a role in immune function. ICF-specific increases in immunoglobulin (Ig) heavy constant mu and delta RNA and cell surface IgM and IgD and decreases in Ig(gamma) and Ig(alpha) RNA and surface IgG and IgA indicate inhibition of the later steps of lymphocyte maturation. ICF-specific increases were seen in RNA for RGS1, a B-cell specific inhibitor of G-protein signaling implicated in negative regulation of B-cell migration, and in RNA for the pro-apoptotic protein kinase C eta gene. ICF-associated decreases were observed in RNAs encoding proteins involved in activation, migration or survival of lymphoid cells, namely, transcription factor negative regulator ID3, the enhancer-binding MEF2C, the iron regulatory transferrin receptor, integrin beta7, the stress protein heme oxygenase and the lymphocyte-specific tumor necrosis factor receptor family members 7 and 17. No differences in promoter methylation were seen between ICF and normal LCLs for three ICF upregulated genes and one downregulated gene by a quantitative methylation assay [combined bisulfite restriction analysis (COBRA)]. Our data suggest that DNMT3B mutations in the ICF syndrome cause lymphogenesis-associated gene dysregulation by indirect effects on gene expression that interfere with normal lymphocyte signaling, maturation and migration.

Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.

We used a proteomic approach to identify proteins that commonly induce an antibody response in lung cancer. Sera from 64 newly diagnosed patients with lung cancer, 99 patients with other types of cancer, and 71 noncancer controls were analyzed for antibody-based reactivity against lung adenocarcinoma proteins resolved by two-dimensional PAGE. Unlike controls, autoantibodies against a protein identified by mass spectrometry as protein gene product 9.5 (PGP 9.5) were detected in sera from 9 of 64 patients with lung cancer. Circulating PGP 9.5 antigen was detected in sera from two additional patients with lung cancer, without detectable PGP 9.5 autoantibodies. PGP 9.5 is a neurospecific polypeptide previously proposed as a marker for non-small cell lung cancer, based on its expression in tumor tissue. Using A549 lung adenocarcinoma cell line, we have demonstrated that PGP 9.5 was present at the cell surface, as well as secreted. Thus, the findings of PGP 9.5 antigen and/or antibodies in serum of patients with lung cancer suggest that PGP 9.5 may have utility in lung cancer screening and diagnosis.

Protein based microarrays: a tool for probing the proteome of cancer cells and tissues.

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.

A database of protein expression in lung cancer.

We have developed a comprehensive approach to identifying molecular changes in lung cancer that includes both genomic and proteomic analyses. The related effort has produced a large amount of data pertaining to gene expression at the RNA and protein levels. As a result, we have constructed a database that contains protein expression data on lung cancer as well as other relevant data including DNA microarray derived data. A large number of proteins that are expressed in different types of lung cancer have been identified and have been correlated with the expression measures for their corresponding genes at the RNA level. The database is intended to facilitate our effort at developing novel classification schemes for lung cancer and the identification of novel markers for early diagnosis.

Identification of androgen-regulated genes in the prostate cancer cell line LNCaP by serial analysis of gene expression and proteomic analysis.

A common therapy for nonorgan-confined prostate cancer involves androgen deprivation. To develop a better understanding of the effect of androgen on prostatic cells, we have analyzed gene expression changes induced by dihydrotestosterone (DHT) in the androgen responsive prostate cancer line LNCaP, at both RNA and protein levels. Changes at the RNA level induced by DHT were determined by means of serial analysis of gene expression (SAGE), and protein profiling was done by means of quantitative two-dimensional polyacrylamide gel electrophoresis. Among 123,371 transcripts analyzed, a total of 28,844 distinct SAGE tags were identified representing 16,570 genes. Some 351 genes were significantly affected by DHT treatment at the RNA level (p < 0.05), of which 147 were induced and 204 repressed by androgen. In two independent experiments, the integrated intensity of 32 protein spots increased and 12 decreased at least two-fold in response to androgen, out of a total of 1031 protein spots analyzed. The change in intensity for most of the affected proteins identified could not be predicted based on the level of their corresponding RNA. Our study provides a global assessment of genes regulated by DHT and suggests a need for profiling at both RNA and protein levels for a comprehensive evaluation of patterns of gene expression.

Proteomics-based identification of RS/DJ-1 as a novel circulating tumor antigen in breast cancer.

PURPOSE: We used a proteomics-based approach to identify tumor proteins that elicit a humoral response in breast carcinoma and that may occur as circulating antigens. EXPERIMENTAL DESIGN: The breast cell line SUM-44 was used as a source of tumor cell proteins for two-dimensional PAGE (2-D PAGE) and for Western blot analysis in which individual sera were analyzed for primary antibodies. RESULTS: Sera from 30 newly diagnosed patients with breast cancer were screened for IgG antibodies to tumor cell proteins. Sera from 116 patients with other cancers and from 25 healthy subjects served as controls. Restricted reactivity against a set of three proteins, identified by mass spectrometry as isoforms of a novel oncogenic protein that regulates RNA-protein interaction (designated RS/DJ-1), was observed in four patients with breast cancer, but not in healthy subjects. The identity was further confirmed by Western blotting with specific antibodies. RS/DJ-1 was found to be secreted in the breast cell line SUM-44, which led us to determine whether RS/DJ-1 was found in circulation in breast cancer. Interestingly, unlike in controls, RS/DJ-1 was readily detectable in sera from 37% of newly diagnosed patients with breast cancer. CONCLUSION: The presence of autoantibodies and/or circulating RS/DJ-1 protein in sera from patients with breast cancer may have clinical utility.

Proteomic approaches within the NCI early detection research network for the discovery and identification of cancer biomarkers.

In the postgenome era, proteomics provides a powerful approach for the analysis of normal and transformed cell functions, for the identification of disease-specific targets, and for uncovering novel endpoints for the evaluation of chemoprevention agents and drug toxicity. Unfortunately, the genomic information that has greatly expounded the genetic basis of cancer does not allow an accurate prediction of what is actually occurring at the protein level within a given cell type at any given time. The gene expression program of a given cell is affected by numerous factors in the in vivo environment resulting from tissue complexity and organ system orchestration, with cells acting in concert with each other and responding to changes in their microenvironment. Repositories of genomic information can be considered master "inventory lists" of genes and their maps, which need to be supplemented with protein-derived information. The National Cancer Institute's Early Detection Research Network is employing proteomics, or "protein walking", in the discovery and evaluation of biomarkers for cancer detection and for the identification of high-risk subjects. Armed with microdissection techniques, including the use of Laser Capture Microdissection (LCM) to procure pure populations of cells directly from human tissue, the Network is facilitating the development of technologies that can overcome the problem of tissue heterogeneity and address the need to identify markers in easily accessible biological fluids. Proteomic approaches complement plasma-based assays of circulating DNA for cancer detection and risk assessment. LCM, coupled with downstream proteomics applications, such as two-dimensional polyacrylamide gel electrophoresis and SELDI (surface enhanced laser desorption ionization) separation followed by mass spectrometry (MS) analysis, may greatly facilitate the characterization and identification of protein expression changes that track normal and disease phenotypes. We highlight recent work from Network investigators to demonstrate the potential of proteomics to identify proteins present in cancer tissues and body fluids that are relevant for cancer screening.

Organ-specific molecular classification of primary lung, colon, and ovarian adenocarcinomas using gene expression profiles.

Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. The establishment of organ-specific gene expression patterns represents a crucial first step in the clinical application of the molecular approach. Here, we report on the gene expression profiles of 154 primary adenocarcinomas of the lung, colon, and ovary. Using high-density oligonucleotide arrays with 7129 gene probe sets, comprehensive gene expression profiles of 57 lung, 51 colon, and 46 ovary adenocarcinomas were generated and subjected to principle component analysis and to a cross-validated prediction analysis using nearest neighbor classification. These statistical analyses resulted in the classification of 152 of 154 of the adenocarcinomas in an organ-specific manner and identified genes expressed in a putative tissue-specific manner for each tumor type. Furthermore, two tumors were identified, one in the colon group and another in the ovarian group, that did not conform to their respective organ-specific cohorts. Investigation of these outlier tumors by immunohistochemical profiling revealed the ovarian tumor was consistent with a metastatic adenocarcinoma of colonic origin and the colonic tumor was a pleomorphic mesenchymal tumor, probably a leiomyosarcoma, rather than an epithelial tumor. Our results demonstrate the ability of gene expression profiles to classify tumors and suggest that determination of organ-specific gene expression profiles will play a significant role in a wide variety of clinical settings, including molecular diagnosis and classification.

Two messenger RNAs and five isoforms for Po66-CBP, a galectin-8 homolog in a human lung carcinoma cell line.

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.