Novel serum biomarkers for renal cell carcinoma NIH AACR

Background: Renal cell carcinoma (RCC) is often undetected until it is in an advanced stage. Currently there are no reliable blood biomarkers to monitor the presence of the disease and/or response to therapies. Mass spectrometry (MS)-based proteomics provides tools for sensitive and high-throughput screening of proteinaceous biological samples and greatly facilitates biomarker iscovery.

Materials and methods: Sera from patients diagnosed with RCC were obtained at the baseline (n=14), then from the same individuals at 1 week (n=10), 2 weeks (n=5), and 3 weeks (n=7) after radical nephrectomy. In addition, unrelated RCC (n=4) and healthy control sera (n=30) were collected. Levels of inflammatory cytokines and growth factors (interleukins 1β, IL 2-7, 10, 12, and 13, GM-CSF, interferon-γ, TNF-α) were quantified in multiplex immunoassays. SELDI TOF MS protein profiling of the whole sera and specimens immunoprecipitated with anti-kallikrein antibodies was done in IMAC-Cu arrays. Further purification and identification of differentially expressed proteins was achieved by combination of two-dimensional gel electrophoresis (2DPAGE), in-gel trypsin digestion and LC-ESIMS for peptide fingerprinting. LC-ESIMS/MS was then used for de novo peptide sequence analysis. Mass spectral data were reduced using the Mascot database search engine. Initial matches were refined and confirmed against raw data and searched against decoy databases to reinforce identifications. Data were further refined using Progenesis Stats.

Results: Serum levels of all growth factors and cytokines (except IL-6) increased above the baseline until 3 weeks post-nephrectomy, with significant (p<0.05)>

Conclusions: SAA, an inflammatory acute phase protein, has been viewed as a biomarker of host response in many human cancers. The recently reported presence of SAA in tumor tissue suggests alternative function of the protein. Sudden disappearance of SAA versus persistent upregulation of multiple inflammatory proteins in post-nephrectomy RCC sera supports this notion. The additional matches derived from the 2D gels of the immunoprecipitates are all either reasonably expected or supported by recent literature identifications in other pathologies. The identifications of the other targets are being confirmed by independent methods. Selective proteome isolation by targeted MS is a promising analytical tool with potential clinical applications.

Identification of Serum Biomarkers for Lung Cancer

Background: Symptoms of lung cancer (LC) often do not appear until the disease is advanced; only 15% of LC cases are discovered while the tumor is in the early stages of development. Carcinogen exposure, asthma and smoking have been determined to be risk factors for the development of LC. Early detection of LC will likely have a major impact on the natural history of the disease and facilitate curative treatment. The objective of this study was to apply multiplexed immunoassays to identify a panel of biomarkers for early detection of LC.

Methods: Normal (NO) serum controls (n = 30) from healthy volunteers and lung cancer patients (n = 30) were acquired from a commercial vendor. Baseline (pre-treatment) serum specimens from individuals with asthma (AST; n = 28) and lung cancer risk (LCR; n = 73) were available from clinical trials of two novel agents that are being developed by JBNI Inc. for the respective indications. Serum levels of 59 cytokines, growth factors and biologically active peptides were quantified using multiplexed immunoassays using the Luminex platform to identify biomarkers that are expressed in a significantly different manner in individuals with LC, LCR, or AST in comparison with NO subjects. Data were reduced using nearest neighbor cluster analysis with squared Euclidian distance to separate patients into groups across analytes with inter-pathology comparisons determined using Student’s t test.

Results: Multiple analytes showed highly significant differences (p < 0.0002) between LC and healthy controls as single indices of pathologic state. In addition we were able to differentiate AST from LC (p < 0.002) and LCR and LC (p < 0.0001) using a panel of thirteen markers in various combinations. Using multiplexed assays we found significant differences in biomarker levels in sera of LC compared to NO, in NO compared to AST and LC compared to AST samples. Our results support an extended multiplexed immunoassay-based analysis of serum biomarker profiles as supplementary tools for the diagnosis of pathologic and as an aid in the development of novel agents for prevention, early detection and treatment of LC.

Conclusions: We have identified a group of markers having high inter-pathology discrimination power that are capable of reliably differentiating AST and LC from control specimens. This panel remains to be validated in a larger set of specimens but we are confident that these measures will produce clinical assays capable of reliably diagnosing lung pathology.