Exploiting the MUC5AC Antigen for Noninvasive Identification of Pancreatic Cancer.

Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with 89Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. 89Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer 89Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

Significance of Liquid Biopsy for Monitoring and Therapy Decision of Colorectal Cancer.

PURPOSE: Despite therapeutic improvements, all patients with nonresectable metastatic colorectal cancer (mCRC) acquire resistance to treatment probably due to the growth of mutated clones. In contrast to tissue-based studies, liquid biopsies have enabled the opportunity to reveal emerging resistance to treatment by detecting mutated clones and noninvasively monitoring clonal dynamics during therapy. METHODS: The courses of three patients with mCRC who were initially RAS wild-type were monitored longitudinally using liquid biopsy with long-term follow-up of up to 20 sequential samples. Detection of fragmented RAS mutated circulating cell-free DNA (cf)DNA in plasma was performed by BEAMing. In addition, plasma digital droplet PCR was used to detect and quantify BRAF and PIK3CA mutated cfDNA. Changes of mutational load were correlated with imaging data. RESULTS: A combination of liquid biopsy and radiological imaging enabled visualization of the occurrence of clonal redistribution after discontinuation of anti-EGFR mAb therapy, as well as emerging RAS mutations during therapy with anti-EGFR mAb indicating resistance. Furthermore, we found that growth of RAS mutated clones is independent of direct selective pressure by anti-EGFR therapy, which is a significant and new finding of this study. CONCLUSIONS: Our findings demonstrated the whole spectrum of clonal selection and redistribution of mutated cell clones leading to acquired resistance. Given our observation that the growth of RAS mutated clones can evolve even in the absence of anti-EGFR mAb therapy, there is a clear imperative to monitor RAS mutations in serial blood draws in all RAS wild-type patients in general and independent of the therapy.

New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions.

BACKGROUND: Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. RESULTS: Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. CONCLUSION: The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.

Immunoscreening of the extracellular proteome of colorectal cancer cells.

BACKGROUND: The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. METHODS: For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. RESULTS: From 281 secretome proteins stained with autoantibodies in total we first defined the "background patterns" of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunoreactivities. CONCLUSIONS: Our findings suggest, first, that autoantibody responses may be due, in large part, to cross-presentation of antigens to the immune system via exosomes, membrane vesicles released by tumor cells and constituting a significant fraction of the secretome. In addition, this immunosecretomics approach has revealed novel biomarker candidates, some of them secretome-specific, and thus serves as a promising complementary tool to the frequently reported immunoproteomic studies for biomarker discovery.

High-level inducible Smad4-reexpression in the cervical cancer cell line C4-II is associated with a gene expression profile that predicts a preferential role of Smad4 in extracellular matrix composition.

BACKGROUND: Smad4 is a tumour suppressor frequently inactivated in pancreatic and colorectal cancers. We have recently reported loss of Smad4 in every fourth carcinoma of the uterine cervix. Smad4 transmits signals from the TGF-beta superfamily of cytokines and functions as a versatile transcriptional co-modulator. The prevailing view suggests that the tumour suppressor function of Smad4 primarily resides in its capability to mediate TGF-beta growth inhibitory responses. However, accumulating evidence indicates, that the acquisition of TGF-beta resistance and loss of Smad4 may be independent events in the carcinogenic process. Through inducible reexpression of Smad4 in cervical cancer cells we wished to shed more light on this issue and to identify target genes implicated in Smad4 dependent tumor suppression. METHODS: Smad4-deficient human C4-II cervical carcinoma cells were used to establish inducible Smad4 reexpression using the commercial Tet-ontrade mark system (Clontech). The impact of Smad4 reexpression on cell growth was analysed in vitro and in vivo. Transcriptional responses were assessed through profiling on cDNA macroarrays (Clontech) and validated through Northern blotting. RESULTS: Clones were obtained that express Smad4 at widely varying levels from approximately physiological to 50-fold overexpression. Smad4-mediated tumour suppression in vivo was apparent at physiological expression levels as well as in Smad4 overexpressing clones. Smad4 reexpression in a dose-dependent manner was associated with transcriptional induction of the extracellular matrix-associated genes, BigH3, fibronectin and PAI-1, in response to TGF-beta. Smad4-dependent regulation of these secreted Smad4 targets is not restricted to cervical carcinoma cells and was confirmed in pancreatic carcinoma cells reexpressing Smad4 after retroviral transduction and in a stable Smad4 knockdown model. On the other hand, the classical cell cycle-associated TGF-beta target genes, c-myc, p21 and p15, remained unaltered. CONCLUSION: Our results show that Smad4-mediated tumour suppression in cervical cancer cells is not due to restoration of TGF-beta growth inhibitory responses. Rather, tumour cell-ECM interactions may be more relevant for Smad4-mediated tumour suppression. C4-II cells with a high level inducible Smad4 expression may serve as a model to indicate further Smad4 targets responsive to diverse environmental stimuli operative in vivo.