Michael Neumaier

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction.

PURPOSE To establish a sensitive assay for the specific detection of carcinoembryonic antigen (CEA)-expressing tumor cells in the bone marrow of patients with colorectal cancer and other CEA-positive carcinomas. PATIENTS AND METHODS A CEA-specific nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay was developed and optimized using limiting dilutions of a CEA-positive cancer cell line mixed with normal bone marrow cell specimens. The optimized test was then used to examine bone marrow samples obtained from 15 patients with abdominal carcinomas (colorectal, n = 10; pancreatic, n = 3; gastric, n = 2) and six patients with breast cancer. Specificity was assessed by examination of 56 negative controls (malignant hematologic disease, n = 28; nonmalignant disease conditions, n = 5; healthy bone marrow donors, n = 8; normal peripheral-blood samples, n = 15). For 11 patients with abdominal carcinomas, immunostaining evaluations were performed using an anti-CEA and an anticytokeratin antibody, and the results compared with the nested PCR assay. RESULTS In the sensitivity calibration system, single CEA-expressing tumor cells were detected in 2 to 5 x 10(7) normal bone marrow cells. All 56 control samples failed to amplify. This demonstrates that mRNAs coding for highly homologous CEA-related antigens expressed by various lineages of blood cells do not interfere. Bone marrow samples from 10 of 15 patients with abdominal cancers and four of six breast cancer patients scored positive, indicating micrometastatic bone disease. Four of 11 samples from the gastrointestinal cancer patients were found to be positive by the PCR method, but were negative with the immunocytology method. CONCLUSION Since approximately 30% of the colorectal carcinoma patients that score negative in immunocytology staining of bone marrow samples have been reported to relapse, earlier diagnosis of the presence of malignant cells is needed. Our result that samples scoring positive in the described CEA-specific PCR test remained negative by two immunostaining methods suggests a higher sensitivity. We conclude that PCR amplification of CEA mRNA may lead to an earlier diagnosis of micrometastatic bone disease in patients with CEA-expressing carcinomas.

Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers

BACKGROUND Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo. OBJECTIVES To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model. STUDY DESIGN ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts. RESULTS 125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h. CONCLUSION Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter‐ and intra‐assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Diagnosis of micrometastases by the amplification of tissue-specific genes

Micrometastases of solid tumors are most commonly detected by immunocytochemistry using monoclonal antibodies directed against tissue-specific gene products like cytokeratin-18 (CK-18) and the carcinoembryonic antigen (CEA). While CK-18 is a marker for epithelia in general, CEA is mainly employed in the detection of gastrointestinal and breast carcinomas. To improve the sensitivity and specificity of micrometastasis detection, we planned to establish polymerase chain reaction (PCR) assays for both markers. Here we provide strong evidence for the existence of a CK-18 pseudogene, since specific amplification (i) was readily obtained from healthy bone marrow donors, (ii) did not require reverse transcription of CK-18 mRNA and (iii) was not abolished by RNase treatment. Using a CK-18-specific probe, Southern blot analyses revealed identical-size fragments for both genomic DNA and a CK-18 cDNA after digestion with appropriate restriction enzymes. On the other hand, the amplification of CEA mRNA (i) was never observed in bone marrow samples of healthy donors or patients without solid tumors, (ii) required intact mRNA and the reverse transcriptase reaction, and (iii) could not be obtained after RNase treatment. In reconstitution experiments, single CEA-expressing tumor cells were reliably detected among 2 x 10(7) normal bone marrow cells. We conclude that, due to the presence of pseudogene(s), PCR-based detection systems are not readily suitable for CK-18, while the CEA mRNA amplification should provide a sensitive and specific test for the presence of ectopic, and hence presumed malignant, CEA-expressing cells in body fluids.

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.

Cytokine concentration in aqueous humour of eyes with exudative age-related macular degeneration

Purpose:  To measure the concentration of cytokines in the aqueous humour of eyes with exudative age‐related macular degeneration (AMD).

The human tumor suppressor CEACAM1 modulates apoptosis and is implicated in early colorectal tumorigenesis.

Defects in the adenomatous polyposis coli (APC) tumor suppressor pathway are sufficient for neoplastic transformation as the initiating step in colorectal carcinogenesis. In contrast, hyperplastic tumors possess normal APC function, and it is unclear whether they represent significant precursor lesion in cancer development. CEACAM1 is a tumor suppressor whose expression is known to be lost in the great majority of early adenomas and carcinomas. We found that loss of CEACAM1 expression is more common in neoplastic tumors than APC mutations. While APC function was normal in hyperplastic aberrant cypt foci and hyperplastic polyps, loss of CEACAM1 was observed as frequently as in the neoplasias. Moreover, the presence or absence of CEACAM1 expression in the hyperplastic tumors correlates with normal or reduced apoptosis, respectively. In vitro, CEACAM1 acts as a regulator of apoptosis in CEACAM1-transfected Jurkat cells. Finally, in human HT29 colon cancer cells, apoptosis can be specifically restored by induction of CEACAM1 expression. These data suggest an oncodevelopmental link between neoplasia and hyperplasia and demonstrate that CEACAM1 acts as a regulator of apoptosis in the colonic epithelium. Thus, failure of the maturing colon cell to express CEACAM1 is likely to contribute to the development of hyperplastic lesions, which may eventually pave the way to neoplastic transformation and colon cancer development.

Pulmonary embolism: CT signs and cardiac biomarkers for predicting right ventricular dysfunction

The aim of this study was to prospectively evaluate the accuracy of quantitative cardiac computed tomography (CT) parameters and two cardiac biomarkers (N-terminal-pro-brain natriuretic peptide (NT-pro-BNP) and troponin I), alone and in combination, for predicting right ventricular dysfunction (RVD) in patients with acute pulmonary embolism. 557 consecutive patients with suspected pulmonary embolism underwent pulmonary CT angiography. Patients with pulmonary embolism also underwent echocardiography and NT-pro-BNP/troponin I serum level measurements. Three different CT measurements were obtained (right ventricular (RV)/left ventricular (LV)axial, RV/LV4-CH and RV/LVvolume). CT measurements and NT-pro-BNP/troponin I serum levels were correlated with RVD at echocardiography. 77 patients with RVD showed significantly higher RV/LV ratios and NT-pro-BNP/troponin I levels compared to those without RVD (RV/LVaxial 1.68±0.84 versus 1.00±0.21; RV/LV4-CH 1.52±0.45 versus 1.01±0.21; RV/LVvolume 1.97±0.53 versus 1.07±0.52; serum NT-pro-BNP 6,372±2,319 versus 1,032±1,559 ng·L−1; troponin I 0.18±0.41 versus 0.06±0.18 g·L−1). The area under the curve for the detection of RVD of RV/LVaxial, RV/LV4-CH, RV/LVvolume, NT-pro-BNP and troponin I were 0.84, 0.87, 0.93, 0.83 and 0.70 respectively. The combination of biomarkers and RV/LVvolume increased the AUC to 0.95 (RV/LVvolume with NT-pro-BNP) and 0.93 (RV/LVvolume with troponin I). RV/LVvolume is the most accurate CT parameter for identifying patients with RVD. A combination of RV/LVvolume with NT-pro-BNP or troponin I measurements improves the diagnostic accuracy of either test alone.

Cytokine concentration in aqueous humor of eyes with diabetic macular edema.

Purpose: To measure cytokine concentrations in aqueous humor of eyes with diffuse diabetic macular edema. Methods: The interventional clinical comparative study included a study group of 23 patients with diffuse diabetic macular edema and a control group of 22 patients undergoing cataract surgery. Cytokine concentrations were measured in aqueous humor samples using a Luminex xMAP suspension array technology. Results: In the study group as compared with the control group, significantly higher concentrations were measured for epidermal growth factor (P < 0.001), human growth factor (P < 0.001), intercellular adhesion molecule-1 (ICAM-1; P < 0.001), interleukin (IL)-1a2 (P = 0.04), IL-6 (P = 0.001), IL-8 (P < 0.001), interferon gamma–induced protein (P = 0.004), monocyte chemoattractant protein-1 (P < 0.001), monokine induced by interferon gamma (P < 0.001), matrix metalloproteinase 1 (P = 0.02), matrix metalloproteinase 9 (P < 0.001), plasminogen activator inhibitor 1 (P < 0.001), placenta growth factor (P < 0.001), tissue growth factor beta (P = 0.003), vascular cell adhesion molecule (P < 0.001), and vascular endothelial growth factor (P < 0.001). Retinal macula thickness was significantly associated with the concentrations of the epidermal growth factor (P = 0.005; &rgr; = 0.45), ICAM-1 (P < 0.001; &rgr; = 0.65), IL-3 (P = 0.002; &rgr; = 0.48), IL-6 (P = 0.003; &rgr; = 0.47), IL-8 (P < 0.001; &rgr; = 0.71), monocyte chemoattractant protein-1 (P = 0.001; &rgr; = 0.53), monokine induced by interferon gamma (P < 0.001; &rgr; = 0.57), matrix metalloproteinase 9 (P < 0.001; &rgr; = 0.61), tissue growth factor beta (P = 0.01; &rgr; = 0.42), placenta growth factor (P = 0.004; &rgr; = 0.46), vascular cell adhesion molecule (P = 0.006; &rgr; = 0.44), and vascular endothelial growth factor (P = 0.01; &rgr; = 0.42). In multivariate analysis, macular thickness remained to be significantly associated with the concentration of ICAM-1 (P = 0.03; r = 0.30). Vascular endothelial growth factor concentrations were correlated with concentration of placenta growth factor (P < 0.001; &rgr; = 0.78), plasminogen activator inhibitor 1 (P = 0.001; &rgr; = 0.54), ICAM-1 (P < 0.001; &rgr; = 0.47), monokine induced by interferon gamma (P = 0.004; &rgr; = 0.44), monocyte chemoattractant protein-1 (P = 0.003; &rgr; = 0.43), vascular cell adhesion molecule (P = 0.01; &rgr; = 0.38), IL-6 (P = 0.02; &rgr; = 0.35), IL-8 (P = 0.02; &rgr; = 0.37), epidermal growth factor (P = 0.01; &rgr; = 0.39), and macrophage migration inhibitory factor (P = 0.01; &rgr; = 0.37). Conclusion: Numerous cytokines are associated with the presence and the amount of diabetic macular edema. Among these cytokines, ICAM-1 was the most significantly associated with the disease parameters.

Micro-CT Based Experimental Liver Imaging Using a Nanoparticulate Contrast Agent: A Longitudinal Study in Mice

Background Micro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging. Methodology ExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano. Principal Findings After a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks. Conclusions The nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.