Monitoring CSF proteome alterations in amyotrophic lateral sclerosis: obstacles and perspectives in translating a novel marker panel to the clinic.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal disorder of the motor neuron system with poor prognosis and marginal therapeutic options. Current clinical diagnostic criteria are based on electrophysiological examination and exclusion of other ALS-mimicking conditions. Neuroprotective treatments are, however, most promising in early disease stages. Identification of disease-specific CSF biomarkers and associated biochemical pathways is therefore most relevant to monitor disease progression, response to neuroprotective agents and to enable early inclusion of patients into clinical trials. METHODS AND FINDINGS: CSF from 35 patients with ALS diagnosed according to the revised El Escorial criteria and 23 age-matched controls was processed using paramagnetic bead chromatography for protein isolation and subsequently analyzed by MALDI-TOF mass spectrometry. CSF protein profiles were integrated into a Random Forest model constructed from 153 mass peaks. After reducing this peak set to the top 25%, a classifier was built which enabled prediction of ALS with high accuracy, sensitivity and specificity. Further analysis of the identified peptides resulted in a panel of five highly sensitive ALS biomarkers. Upregulation of secreted phosphoprotein 1 in ALS-CSF samples was confirmed by univariate analysis of ELISA and mass spectrometry data. Further quantitative validation of the five biomarkers was achieved in an 80-plex Multiple Reaction Monitoring mass spectrometry assay. CONCLUSIONS: ALS classification based on the CSF biomarker panel proposed in this study could become a valuable predictive tool for early clinical risk stratification. Of the numerous CSF proteins identified, many have putative roles in ALS-related metabolic processes, particularly in chromogranin-mediated secretion signaling pathways. While a stand-alone clinical application of this classifier will only be possible after further validation and a multicenter trial, it could be readily used to complement current ALS diagnostics and might also provide new insights into the pathomechanisms of this disease in the future.

Autoantibodies against the exocrine pancreas in autoimmune pancreatitis: gene and protein expression profiling and immunoassays identify pancreatic enzymes as a major target of the inflammatory process.

OBJECTIVES: Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP. METHODS: To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis. RESULTS: Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen. CONCLUSIONS: These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.

Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is characterized by aggressive tumor behavior coupled with poor prognosis. Various etiologies have been linked to HCC development, most prominently chronic hepatitis B and C virus infections as well as chronic alcohol consumption. In approximately 10% of HCCs, the etiology remains cryptic; however, recent epidemiological data suggest that most of these cryptogenic HCCs develop due to nonalcoholic steatohepatitis. To identify etiology-dependent DNA copy number aberrations and genes relevant to hepatocarcinogenesis, we performed array-based comparative genomic hybridization of 63 HCCs of well-defined etiology and 4 HCC cell lines followed by gene expression profiling and functional analyses of candidate genes. For a 10-megabase chromosome region on 8q24, we observed etiology-dependent copy number gains and MYC overexpression in viral and alcohol-related HCCs, resulting in up-regulation of MYC target genes. Cryptogenic HCCs showed neither 8q24 gains, nor MYC overexpression, nor target gene activation, suggesting that tumors of this etiology develop by way of a distinct MYC-independent pathomechanism. Furthermore, we detected several etiology-independent small chromosome aberrations, including amplification of MDM4 on 1q32.1 and frequent gains of EEF1A2 on 20q13.33. Both genes were overexpressed in approximately half the HCCs examined, and gene silencing reduced cell viability as well as proliferation and increased apoptosis rates in HCC cell lines. CONCLUSION: Our findings suggest that MDM4 and EEF1A2 act as etiology-independent oncogenes in a significant percentage of HCCs.

Array-based analysis of genomic DNA methylation patterns of the tumour suppressor gene p16INK4A promoter in colon carcinoma cell lines.

Aberrant DNA methylation at CpG dinucleotides can result in epigenetic silencing of tumour suppressor genes and represents one of the earliest events in tumourigenesis. To date, however, high-throughput tools that are capable of surveying the methylation status of multiple gene promoters have been restricted to a limited number of cytosines. Here, we present an oligonucleotide microarray that permits the parallel analysis of the methylation status of individual cytosines, thus combining high throughput and high resolution. The approach was used to study the CpG island in the promoter region of the tumour suppressor gene p16(INK4A). In total, 876 oligonucleotide probes of 21 nt in length were used to inspect the methylation status of 53 CpG dinucleotides, producing correct signals in colorectal cancer cell lines as well as control samples with a defined methylation status. The information was validated by established alternative methods. The overall methylation pattern was consistent for each cell line, while different between them. At the level of individual cytosines, however, significant variations between individual cells of the same type were found, but also consistencies across the panel of cancer cell lines were observed.