Colon-derived liver metastasis, colorectal carcinoma, and hepatocellular carcinoma can be discriminated by the Ca(2+)-binding proteins S100A6 and S100A11.

BACKGROUND: It is unknown, on the proteomic level, whether the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, tissue from colon-derived liver metastases (n = 17) were classified, laser-microdissected, and analysed by ProteinChip arrays (SELDI). The resulting spectra were compared with data for primary colorectal (CRC) and hepatocellular carcinomas (HCC) from our former studies. Of 49 signals differentially expressed in primary HCC, primary CRC, and liver metastases, two were identified by immunodepletion as S100A6 and S100A11. Both proteins were precisely localized immunohistochemically in cells. S100A6 and S100A11 can discriminate significantly between the two primary tumor entities, CRC and HCC, whereas S100A6 allows the discrimination of metastases and HCC. CONCLUSIONS: Both identified proteins can be used to discriminate different tumor entities. Specific markers or proteomic patterns for the metastases of different primary cancers will allow us to determine the biological characteristics of metastasis in general. It is unknown how the protein patterns of tumors change during metastasis or whether markers are present that allow metastases to be allocated to a specific tumor entity. The latter is of clinical interest if the primary tumor is not known.

Tapping an unexploited repository: Carnoy's fixed cell pellets for proteomic biomarker research in leukemia.

For chromosomal analysis in tumor genetics, cells from blood and bone marrow are prepared and preserved virtually indefinitely in Carnoy's fixative (methanol/acetic acid). Numerous samples are stored unvalued in hospitals and institutes worldwide. We developed a method to analyze proteins from even a small amount of these cells by mass spectrometry using affinity chromatographic surfaces (SELDI), and demonstrated the application of proteomic biomarker research in cases of acute myeloid leukemia.

Specific pattern of protein expression in acute myeloid leukemia harboring FLT3-ITD mutations.

FLT3 activating mutations can be detected in about 35% of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) represent the majority of FLT3 mutations (25 - 30%) while FLT3-TKD (tyrosine kinase domain) mutations can be found in about 7% of AML patients. In this study, we addressed the question whether especially primary AML cells carrying FLT3-ITD mutations show differences in terms of their protein expression pattern compared to FLT3 wild-type blasts. We investigated bone marrow samples that were isolated at diagnosis from 36 AML patients expressing either FLT3 wild-type (n = 16) or an activating FLT3 mutation (FLT3-ITD, n = 15; FLT3-TKD, n = 5). Proteomic analysis was performed by means of surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry which has shown its high efficiency in finding biomarkers in solid tumors. Here, we demonstrate that a large series of proteins is differently expressed in primary AML blasts harboring FLT3-ITD mutations. Furthermore, there are also significant differences of the protein expression profile between FLT3-ITD and FLT3-TKD mutations. Interestingly, further analysis of FLT3-ITD positive AML according to its response to the induction chemotherapy demonstrates putative prognostic markers for this subgroup of AML. We suggest that SELDI-TOF mass spectrometry represents a promising tool of proteomic analysis of AML that might help to establish new prognostic markers in AML.

Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker.

BACKGROUND: Patients with pancreatic adenocarcinomas have a poor prognosis because of late clinical manifestation and the tumor's aggressive nature. We used proteomic techniques to search for markers of pancreatic carcinoma. METHODS: We performed protein profiling of microdissected cryostat sections of 9 pancreatic adenocarcinomas and 10 healthy pancreatic tissue samples using ProteinChip technology (surface-enhanced laser desorption/ionization). We identified proteins by use of 2-dimensional gel electrophoresis, peptide fingerprint mapping, and immunodepletion and used immunohistochemistry for in situ localization of the proteins found. We used ELISA to quantify these proteins in preoperative serum samples from 35 patients with pancreatic cancer and 37 healthy individuals. RESULTS: From among the differentially expressed signals that were detected by ProteinChip technology, we identified 2 proteins, DJ-1 and heat shock protein 27 (HSP27). We then detected HSP27 in sera of patients by use of ELISA, indicating a sensitivity of 100% and a specificity of 84% for the recognition of pancreatic cancer. CONCLUSIONS: The detection of DJ-1 and HSP27 in pure defined tissue and the retrieval of HSP27 in serum by antibody-based methods identifies a potential marker for pancreatic cancer.

Identification of specific protein markers in microdissected hepatocellular carcinoma.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers.

Protein profiling of oral brush biopsies: S100A8 and S100A9 can differentiate between normal, premalignant, and tumor cells.

In oral mucosa lesions it is frequently difficult to differentiate between precursor lesions and already manifest oral squamous cell carcinoma. Therefore, multiple scalpel biopsies are necessary to detect tumor cells already in early stages and to guarantee an accurate follow-up. We analyzed oral brush biopsies (n = 49) of normal mucosa, inflammatory and hyperproliferative lesions, and oral squamous cell carcinoma with ProteinChip Arrays (SELDI) as a non-invasive method to characterize putative tumor cells. Three proteins were found that differentiated between these three stages. These three proteins are able to distinguish between normal cells and tumor cells with a sensitivity of 100% and specificity of 91% and can distinguish inflammatory/hyperproliferative lesions from tumor cells with a sensitivity of up to 91% and specificity of up to 90%. Two of these proteins have been identified by immunodepletion as S100A8 and S100A9 and this identification was confirmed by immunocytochemistry. For the first time, brush biopsies have been successfully used for proteomic biomarker discovery. The identified protein markers are highly specific for the distinction of the three analyzed stages and therewith reflect the progression from normal to premalignant non-dysplastic and finally to tumor tissue. This knowledge could be used as a first diagnostic step in the monitoring of mucosal lesions.

Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling.

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.

Protein profiles of bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis.

BACKGROUND: Pulmonary sarcoidosis is a multisystem granulomatous disease with various clinical phenotypes. So far, there has been little information on protein patterns (PPs) of bronchoalveolar lavage fluid (BALF) from patients with sarcoidosis and no data are available on PPs in clinical disease subtypes. OBJECTIVES: To investigate the PP of BALF from patients with pulmonary sarcoidosis, to evaluate whether PPs reflect disease course as assessed by chest X-ray (CXR), and to compare PPs between patients with/without Löfgren's syndrome. METHODS: Surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy was applied to investigate PPs in unconcentrated BALF from 65 patients (CXR stage I, n = 32; CXR stage II, n = 22, CXR stage III, n = 11) and 23 healthy control subjects. The Mann-Whitney U test was used to detect differentially expressed protein peaks. After reversed-phase fractionation, peptide fingerprint mapping and immunodepletion were used to identify deregulated (up-regulated or down-regulated) proteins. RESULTS: Forty differentially expressed protein entities (2.75-185.62 kD) were detected in patients with pulmonary sarcoidosis versus control subjects (p < 0.05). Whereas 13 peaks (33%) were present across all CXR stages, 27 (67%) were specific for particular CXR stages. Comparison of PPs between CXR stage I patients with or without Löfgren's syndrome revealed 25 differentially expressed peaks. The total number of deregulated peaks and also of those associated with sarcoidosis as a whole were markedly lower in patients with Löfgren's syndrome in comparison with other sarcoid phenotypes. Human serum albumin, alpha1-antitrypsin, and protocadherin-2 precursor were identified from sarcoidosis-associated PP. CONCLUSION: Surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy enables determination of protein patterns in sarcoid BALF and allows detection of protein patterns linked to a particular disease course.

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.

Different expression of calgizzarin (S100A11) in normal colonic epithelium, adenoma and colorectal carcinoma.

The aim of the study was to detect proteomic markers usable to distinguish colorectal carcinoma from colon adenoma for a better understanding of the molecular mechanisms in the process of tumourigenesis. Therefore, we microdissected colon carcinoma tissue, epithelial colon adenoma tissue as well as normal adjacent colon epithelium and determined protein profiles by SELDI-TOF MS. A multitude of significantly different signals was detected. For their identification colon biopsis were lysed and subjected to a two-dimensional gel electrophoresis for separation. Subsequently, we identified nearly 100 proteins by tryptic digestion, peptide fingerprint mapping and database search. Calgizzarin (S100A11; S100C) identified by peptide fingerprint mapping correlated very well with a significantly differentially expressed signal found in prior protein profiling. Using an immunodepletion assay we confirmed the identity of this signal as calgizzarin. To localise calgizzarin in tissues we performed immunohistochemistry. For further confirmation of the identity of calgizzarin we re-analysed IHC-positive as well as IHC-negative tissue sections on ProteinChip arrays. This work demonstrates that biomarkers in colorectal cancer can be detected, identified and assessed by a proteomic approach comprising tissue-microdissection, protein profiling and immunological techniques.

Identification of HNP3 as a tumour marker in CD4+ and CD4- lymphocytes of patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas (CTCL) are characterized by malignant proliferation of skin homing T-cells. Although prognosis is generally good, reliable markers are needed to identify patients at risk for a more aggressive course. ProteinChip (SELDI) technology was used as a tool for the discovery of protein patterns in lymphocytes from patients with CTCL (n=25) and unaffected controls (n=25). Lymphocytes were separated in CD4+ and CD4- fractions by magnetic cell sorting (MACS). Each whole protein extract was analysed by ProteinChip technology. The resulting protein profiles were submitted for bioinformatic analysis including a clustering algorithm, a rule extraction, a rating and a rule-base construction step. For the generated combined rule base for the CD4- cell fraction, both the sensitivity and specificity for the prediction of CTCL reached 96%, while for the CD4+ fraction they were 92% and 84%, respectively, for sensitivity and specificity. The most significant peak at 3489Da could be identified as HNP3, an alpha-defensin, by immunocapturing. These results open up both the possibility for the use of this protein signature, especially HNP3, to more effectively monitor and screen CTCL, and the avenue to identify the other relevant peaks for a better understanding of the development of this tumour.

Characterization of pepsinogen C as a potential biomarker for gastric cancer using a histo-proteomic approach.

We analyzed 74 cryostat sections of central gastric tumor, tumor margin, and normal gastric epithelium using ProteinChip Arrays and SELDI-TOF MS. One peak was significantly down-regulated in tumor tissue (P = 1.43 x 10(-6)) and identified as pepsinogen C using MS/MS analysis and immunodepletion. This signal was further characterized by immunohistochemistry. This work demonstrates that differentially expressed signals can be identified and assessed using a proteomic approach comprising tissue-microdissection, protein profiling, and immunohistochemistry.

Discovery and identification of alpha-defensins as low abundant, tumor-derived serum markers in colorectal cancer.

BACKGROUND & AIMS: Although colorectal cancer is one of the best characterized tumors with regard to the multistep genetic progression, it remains one of the most frequent and deadly neoplasms in Western countries. This is mainly due to the fact that, up to now, no clinically relevant serum markers could be established in an early routine diagnostic procedure. METHODS: We comparatively analyzed microdissected normal and tumorous colonic epithelium by ProteinChip technology to detect proteins specific for the tumor directly in the tissue. Immunohistochemistry (IHC) was used for the in situ localization of the discovered proteins, and an ELISA was performed to quantify these proteins in serum. RESULTS: By this approach, we found and identified alpha-defensins 1-3 (HNP1-3) to be more highly expressed in the tumor than in normal epithelium. These findings could be confirmed by IHC. Detection of these peptides in the corresponding serum samples was subsequently performed with ELISA, resulting in an average sensitivity of 69% and specificity of 100% for the recognition of colorectal cancer when using the HNP1-3 level in the serum of the patients. CONCLUSIONS: The direct analysis of microdissected tissue for the discovery of tumor-specific markers followed by the specific detection of these markers in serum by antibody-based methods proved to be a successful strategy in this study. Therefore, we can conclude that these promising markers would not have been found in serum without the information gained through the analysis of microdissected tissue by ProteinChip technology.

Identification of proteins from colorectal cancer tissue by two-dimensional gel electrophoresis and SELDI mass spectrometry.

We investigated protein profiles obtained from colorectal tumor tissue and adjacent normal mucosa to identify tumor specific changes. Protein extracts of biopsis were separated by two-dimensional gel electrophoresis and >40 low-molecular mass proteins were identified by peptide fingerprinting using surface-enhanced laser desoption/ionization mass spectrometry (SELDI-MS). Among these, PACAP protein, hnrnp A1, flavin reductase, calgizzarin, NDK B (NM23-H2), cyclophilin A and smooth muscle protein 22-alpha showed significantly differential abundancy in the analyzed specimens. In addition, immunohistochemical analysis of tissue distribution and subcellular localization of some of the differentially expressed proteins demonstrated alterations in subcellular protein distribution. Further investigations are in progress to assess whether these differentially expressed proteins are associated with tumor development and tumor progression.

A technical triade for proteomic identification and characterization of cancer biomarkers.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

Proteomic profiling in microdissected hepatocellular carcinoma tissue using ProteinChip technology.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well understood. It is known, however, that cancer development and progression are accompanied by profound changes at the cellular and subcellular level, involving RNA/DNA and protein structure and function. Therefore, high-throughput, proteomic techniques targeting these biological molecules may provide novel insights into HCC genesis and prognosis. We characterized tissue protein profiles from 10 HCC patients using ProteinChip technology (SELDI) which is able to detect minute amounts of proteins and moreover to analyze complex protein pattern. Therefore, after histopathological examination, proteins from kryostat sections of non-tumorous hepatic tissue as well as from central and peripheral tumor areas were isolated from complete histological sections or from selected and microdissected tissue areas. Analysis on the SAX and WCX ProteinChip Arrays revealed 14-26, and 25-29 differentially expressed peaks respectively, which characterized non-tumorous and tumor tissue (p< or =0.05). One feature which allows differentiation between central tumor and peripheral tumor regions could only be detected in microdissected tissue. Using ProteinChip technology in combination with tissue microdissection it is possible to investigate complex changes at the protein level in hepatocellular cancer associated with tumor development and progression.

Biomarker discovery and identification in laser microdissected head and neck squamous cell carcinoma with ProteinChip technology, two-dimensional gel electrophoresis, tandem mass spectrometry, and immunohistochemistry.

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression.