Patterns of Peripheral Blood B-Cell Subtypes Are Associated With Treatment Response in Patients Treated With Immune Checkpoint Inhibitors: A Prospective Longitudinal Pan-Cancer Study.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized systemic anti-tumor treatments across different types of cancer. Nevertheless, predictive biomarkers regarding treatment response are not routinely established yet. Apart from T-lymphocytes, the humoral immunity of B-lymphocytes is studied to a substantially lesser extent in the respective setting. Thus, the aim of this study was to evaluate peripheral blood B-cell subtypes as potential predictors of ICI treatment response. Methods: Thirty-nine cancer patients receiving ICI therapy were included into this prospective single-center cohort study. All had a first blood draw at the date before treatment initiation and a second at the time of first response evaluation (after 8-12 weeks). Seven different B-cell subtypes were quantified by fluorescence-activated cell sorting (FACS). Disease control- (DCR) and objective response rate (ORR) were co-primary study endpoints. Results: Overall, DCR was 48.7% and ORR was 25.6%, respectively. At baseline, there was no significant association of any B-cell subtype with neither DCR nor ORR. At the first response evaluation, an increase in the frequency of CD21- B-cells was a statistically significant negative predictor of response, both regarding DCR (OR=0.05, 95%CI=0.00-0.67, p=0.024) and ORR (OR=0.09, 95%CI=0.01-0.96, p=0.046). An increase of the frequency of switched memory B-cells was significantly associated with reduced odds for DCR (OR=0.06, 95%CI=0.01-0.70, p=0.025). Patients with an increased frequency of naïve B-cells were more likely to benefit from ICI therapy as indicated by an improved DCR (OR=12.31, 95%CI=1.13-134.22, p=0.039). Conclusion: In this study, certain B-cell subpopulations were associated with ICI treatment response in various human cancer types.

Evaluation of autoantibodies as predictors of treatment response and immune-related adverse events during the treatment with immune checkpoint inhibitors: A prospective longitudinal pan-cancer study.

BACKGROUND: The presence of autoantibodies in the serum of cancer patients has been associated with immune-checkpoint inhibitor (ICI) therapy response and immune-related adverse events (irAEs). A prospective evaluation of different autoantibodies in different cancer entities is missing. MATERIALS AND METHODS: In this prospective cohort study, we included a pan-cancer cohort of patients undergoing ICI treatment and measured a comprehensive panel of autoantibodies at treatment start and at the time point of first response evaluation. The presence and induction of autoantibodies (ANA, ENA, myositis, hepatopathy, rheumatoid arthritis) in different cancer entities were assessed and the association between autoantibodies and disease control rate (DCR), objective response rate (ORR), and progression-free survival (PFS), as well as the development of grade 3 or higher irAEs were evaluated by logistic regression models, cox proportional hazard models, and Kaplan-Meier estimators. RESULTS: Of 44 patients with various cancer entities, neither the presence of any positive autoantibody measurement nor the presence of positive antinuclear antibodies (ANA) [≥1:80] at baseline was associated with the examined clinical endpoints (DCR, ORR, PFS) in univariable and multivariable analyses. After 8-12 weeks of ICI treatment, DCR, ORR, and PFS did not significantly differ between patients with and without any positive autoantibody measurement or positive ANA titers. The frequency of irAEs did not differ depending on autoantibody status of the patients. CONCLUSION: Autoantibodies at treatment initiation or induction after 8-12 weeks of ICI treatment are not associated with treatment efficacy as indicated by DCR, ORR, and PFS or higher grade irAEs.

Chronic Inflammation Might Protect Hemodialysis Patients From Severe COVID-19.

Hemodialysis patients (HD) are expected to have excess mortality in coronavirus disease 2019 (COVID-19). This was challenged by a recent study reporting HD patients to have comparable mortality and less ICU admissions when hospitalized with COVID-19. An altered immune system due to chronic inflammation might protect HD-patients from severe COVID-19. Therefore, we aimed to describe the peripheral blood immune phenotype in HD-patients and respective controls with COVID-19. Methods: Sixty-four patients (31 HD, 33 non-HD) with PCR-confirmed COVID-19 and 16 control patients (10 HD, 6 non-HD) were prospectively included. According to symptoms, COVID-19 patients were categorized as asymptomatic/mild, moderate or severe COVID-19 phenotypes. Cytokine profiling and immune phenotyping was performed. Results: Th1 and Th17 plasma cytokine levels were highly increased in HD patients without COVID-19 and were not significantly regulated during COVID-19. In non-HD COVID-19 patients these cytokines increased significantly with disease severity. While all patients with moderate or severe COVID-19 showed hallmarks of COVID-19 such as decreased CD3+, CD4+ and CD8+ and CD4+CD25hiFoxP3+ regulatory T cells, significantly increased CD38+CD8+ effector memory and CD38+CD8+ TEMRA T cells were detected in moderate/severe COVID-19 HD patients, which was not observed in non-HD patients with moderate or severe COVID-19. Furthermore, CD161+CD8+ T cells decreased significantly in non-HD COVID-19 patients dependent on disease severity, but not in HD patients. Dynamics of B cells and subtypes were comparable in HD and non-HD COVID-19 patients. Conclusions: HD patients might be protected from severe COVID-19 due to their chronic inflammatory state with increased CD38+CD8+ effector memory and TEMRA T cells as well as CD161+CD8+ T cells.

Chronic Oxidative Stress Promotes Molecular Changes Associated with Epithelial Mesenchymal Transition, NRF2, and Breast Cancer Stem Cell Phenotype.

Oxidative stress plays a role in carcinogenesis, but it also contributes to the modulation of tumor cells and microenvironment caused by chemotherapeutics. One of the consequences of oxidative stress is lipid peroxidation, which can, through reactive aldehydes such as 4-hydroxy-2-nonenal (HNE), affect cell signaling pathways. On the other hand, cancer stem cells (CSC) are now recognized as a major factor of malignancy by causing metastasis, relapse, and therapy resistance. Here, we evaluated whether oxidative stress and HNE modulation of the microenvironment can influence CSC growth, modifications of the epithelial to mesenchymal transition (EMT) markers, the antioxidant system, and the frequency of breast cancer stem cells (BCSC). Our results showed that oxidative changes in the microenvironment of BCSC and particularly chronic oxidative stress caused changes in the proliferation and growth of breast cancer cells. In addition, changes associated with EMT, increase in glutathione (GSH) and Nuclear factor erythroid 2-related factor 2 (NRF2) were observed in breast cancer cells grown on HNE pretreated collagen and under chronic oxidative stress. Our results suggest that chronic oxidative stress can be a bidirectional modulator of BCSC fate. Low levels of HNE can increase differentiation markers in BCSC, while higher levels increased GSH and NRF2 as well as certain EMT markers, thereby increasing therapy resistance.

GIRK1 triggers multiple cancer-related pathways in the benign mammary epithelial cell line MCF10A.

Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10AGIRK1 with the control (MCF10AeGFP). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

MiR-1287-5p inhibits triple negative breast cancer growth by interaction with phosphoinositide 3-kinase CB, thereby sensitizing cells for PI3Kinase inhibitors.

BACKGROUND: Non-coding RNAs and especially microRNAs have been discovered to act as master regulators of cancer initiation and progression. The aim of our study was to discover and characterize the function of yet functionally uncharacterized microRNAs in human breast carcinogenesis. METHODS: In an unbiased approach, we utilized an established model system for breast cancer (BC) stem cell formation ("mammosphere assay") to identify whole miRNome alterations in breast carcinogenesis. Clinical samples of BC patients were used to evaluate the human relevance of the newly identified miRNA candidates. One promising candidate, miR-1287-5p, was further explored on its impact on several hallmarks of cancer. The molecular mode of action was characterized by whole transcriptome analysis, in silico prediction tools, miRNA-interaction assays, pheno-copy assays, and drug sensitivity assays. RESULTS: Among several other microRNAs, miR-1287-5p was significantly downregulated in mammospheres and human BC tissue compared to normal breast tissue (p < 0.0001). Low expression levels were significantly associated with poor prognosis in BC patients. MiR-1287-5p significantly decreased cellular growth, cells in S phase of cell cycle, anchorage-independent growth, and tumor formation in vivo. In addition, we identified PIK3CB as a direct molecular interactor of miR-1287-5p and a novel prognostic factor in BC. Finally, PI3Kinase pathway chemical inhibitors combined with miR-1287-5p mimic increased the pharmacological growth inhibitory potential in triple negative BC cells. CONCLUSION: Our data identified for the first time the involvement of miR-1287-5p in human BC and suggest a potential for therapeutic interventions in difficult to treat triple negative BC.

A Pilot Randomized Trial Assessing the Effect of a Psychoeducational Intervention on Psychoneuroimmunological Parameters Among Patients With Nonmetastatic Breast Cancer.

OBJECTIVE: The aim of this study was to determine a potential benefit of the specific psychoeducational intervention "Learning to Live with Cancer" (LTLWC) for patients with operated nonmetastatic breast cancer, with respect to psychological variables and endocrine and immune parameters. METHODS: Fifty-two postmenopausal women with operated stage I to III breast cancer were randomized to either a breast cancer intervention group (BCIG, n = 30) who immediately began participating in the LTLWC intervention program or to a breast cancer control group (BCCG, n = 22). Matched healthy women were asked to participate as a noncancer comparison group (n = 26). All participants were evaluated at three different time points (t1-t3) using a set of standardized questionnaires and blood samples were taken to analyze immune cell subsets and stress hormone levels. RESULTS: A significant reduction in trait anxiety/State Trait Anxiety Inventory score was observed in the BCIG (t1: median = 35.0 [interquartile range = 28.0-38.0] versus t3: median = 26.0 [interquartile range = 18.5-37.0], p = .0001) compared with the BCCG (t1: median = 41.0 [interquartile range =32.75-49.0]; t3: median = 38.5 [interquartile range = 30.75-46.5], p = .01524; p interaction = .001). In parallel, a significant rise of serotonin levels (t1: median = 66.5 ng/ml [interquartile range = 11.50-106.00] versus t3: median = 80.5 ng/ml [interquartile range =59.00-118.00], p = .00008) as well as a significant reduction of the elevated number of Treg cells at baseline (t1: median = 4.45% [interquartile range = 4.00-5.33] versus t3: median = 2.80% [interquartile range = 2.68-3.13], p < .00001) were observed in the BCIG versus no change in the BCCG. A significant statistical association between reduced trait anxiety and decreased Treg cell number could be demonstrated in the BCIG (r = .62, p < .01). CONCLUSIONS: The observed results of this study provide preliminary support for the efficacy of the LTLWC program in significantly improving psychoneuroimmunological parameters in patients with nonmetastatic breast cancer.

Immune cell landscape in therapy-naïve squamous cell and adenocarcinomas of the lung.

Squamous cell and adenocarcinomas of the lung develop different mechanisms during carcinogenesis to evade attacks of the immune system. Besides the well-known check-point control programmed death 1 and its ligand, many more mechanisms, acting either tumoricidal or in favor of tumor progression, exist. Analysis of the immune cell profiles in resected tissues and bronchoalveolar lavage samples and correlation between them and with overall survival data was performed. In all tumor samples in this study, cells of the immune system expressed a tumor-cooperating phenotype. High numbers of regulatory T cells, or alternatively expression of Vista on lymphocytes was present. Tumoricidal dendritic cells were absent in tumor tissue, and barely present in bronchoalveolar lavage, whereas tumor-friendly monocytoid and plasmocytoid dendritic cells were seen in both. Alveolar macrophages were predominantly differentiated into tumor-cooperating M2 types, whereas tumoricidal M1 macrophages were absent or rare. The expression of PDL1 on tumor cells did not correlate with any other immune cells. Expression of PD1 on lymphocytes was frequently encountered. None of analyzed immune cells showed correlation with overall survival. Immune cells in bronchoalveolar lavage and tissue did not correlate. For the first time, a tissue-based analysis of different immune cells in squamous cell and adenocarcinomas of the lung is provided, trying to explain their potential role in tumor development and progression. Discordant numbers of cells with bronchoalveolar lavage are most probably due to the fact that bronchoalveolar lavage reflects the situation in the whole lung, where chronic obstructive lung disease and other conditions are present.

Primary patient-derived lung adenocarcinoma cell culture challenges the association of cancer stem cells with epithelial-to-mesenchymal transition.

The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications.

Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.

Comprehensive Analysis of miRNome Alterations in Response to Sorafenib Treatment in Colorectal Cancer Cells.

MicroRNAs (miRNAs) are master regulators of drug resistance and have been previously proposed as potential biomarkers for the prediction of therapeutic response in colorectal cancer (CRC). Sorafenib, a multi-kinase inhibitor which has been approved for the treatment of liver, renal and thyroid cancer, is currently being studied as a monotherapy in selected molecular subtypes or in combination with other drugs in metastatic CRC. In this study, we explored sorafenib-induced cellular effects in Kirsten rat sarcoma viral oncogene homolog olog (KRAS) wild-type and KRAS-mutated CRC cell lines (Caco-2 and HRT-18), and finally profiled expression changes of specific miRNAs within the miRNome (>1000 human miRNAs) after exposure to sorafenib. Overall, sorafenib induced a time- and dose-dependent growth-inhibitory effect through S-phase cell cycle arrest in KRAS wild-type and KRAS-mutated CRC cells. In HRT-18 cells, two human miRNAs (hsa-miR-597 and hsa-miR-720) and two small RNAs (SNORD 13 and hsa-miR-3182) were identified as specifically sorafenib-induced. In Caco-2 cells, nine human miRNAs (hsa-miR-3142, hsa-miR-20a, hsa-miR-4301, hsa-miR-1290, hsa-miR-4286, hsa-miR-3182, hsa-miR-3142, hsa-miR-1246 and hsa-miR-720) were identified to be differentially regulated post sorafenib treatment. In conclusion, we confirmed sorafenib as a potential anti-neoplastic treatment strategy for CRC cells by demonstrating a growth-inhibitory and cell cycle-arresting effect of this drug. Changes in the miRNome indicate that some specific miRNAs might be relevant as indicators for sorafenib response, drug resistance and potential targets for combinatorial miRNA-based drug strategies.

Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer Patients.

Extensive stromal interaction is one reason for the dismal outcome of biliary tract cancer (BTC) patients. Epithelial to mesenchymal transition (EMT) is involved in tumor invasion and metastasis and is partly regulated by microRNAs (miRs). This study explores the expression of anti-EMT miR200 family (miR141, -200a/b/c, -429) and miR205 as well as the EMT-related proteins E-cadherin and vimentin in a panel of BTC cell lines and clinical specimens by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry, respectively. MicroRNA expression was correlated to (i) the expression patterns of E-cadherin and vimentin; (ii) clinicopathological characteristics; and (iii) survival data. MicroRNA-200 family and miR205 were expressed in all BTC cells and clinical specimens. E-cadherin and vimentin showed a mutually exclusive expression pattern in both, in vitro and in vivo. Expression of miR200 family members positively correlated with E-cadherin and negatively with vimentin expression in BTC cells and specimens. High expression of miR200 family members (but not miR205) and E-cadherin was associated with longer survival, while low miR200 family and high vimentin expression was a predictor of unfavorable survival. Overall, the current study demonstrates the relevance of the miR200 family in EMT of BTC tumors and suggests these miRs as predictors for positive outcome.

Genetic and epigenetic analysis of putative breast cancer stem cell models.

BACKGROUND: Cancer stem cell model hypothesizes existence of a small proportion of tumor cells capable of sustaining tumor formation, self-renewal and differentiation. In breast cancer, these cells were found to be associated with CD44⁺CD24-low and ALDH⁺ phenotype. Our study was performed to evaluate the suitability of current approaches for breast cancer stem cell analyses to evaluate heterogeneity of breast cancer cells through their extensive genetic and epigenetic characterization. METHODS: Breast cancer cell lines MCF7 and SUM159 were cultured in adherent conditions and as mammospheres. Flow cytometry sorting for CD44, CD24 and ALDH was performed. Sorted and unsorted populations, mammospheres and adherent cell cultures were subjected to DNA profiling by array CGH and methylation profiling by Epitect Methyl qPCR array. Methylation status of selected genes was further evaluated by pyrosequencing. Functional impact of methylation was evaluated by mRNA analysis for selected genes. RESULTS: Array CGH did not reveal any genomic differences. In contrast, putative breast cancer stem cells showed altered methylation levels of several genes compared to parental tumor cells. CONCLUSIONS: Our results underpin the hypothesis that epigenetic mechanisms seem to play a major role in the regulation of CSCs. However, it is also clear that more efficient methods for CSC enrichment are needed. This work underscores requirement of additional approaches to reveal heterogeneity within breast cancer.

Effect of oxidative stress and endotoxin on human serum albumin in brain-dead organ donors.

Albumin, among other molecules, binds and detoxifies endotoxin in healthy people. Oxidative stress leads to protein oxidation and thus to the impaired binding properties of albumin. This property, in combination with increased gut permeability, leads to the appearance of endotoxin in the systemic circulation and to impaired organ function. We hypothesize that these processes occur in the serum of brain-dead organ donors. Endotoxin was determined with an adapted Limulus amoebocyte lysate assay. The albumin fractions and binding capacity were determined by high-performance liquid chromatography (HPLC). FlowCytomix (eBioscience, San Diego, Calif) was used to determine the cytokine levels. Carbonylated proteins (CPs) and myeloperoxidase (MPO) were measured by an enzyme-linked immunosorbent assay (ELISA). Eighty-four brain-dead organ donors were enrolled and categorized by the duration of intensive care unit (ICU) stay. The albumin-binding capacity for dansylsarcosine was reduced in brain-dead patients compared with controls. Endotoxin positivity in 16.7% of donors was associated with decreased binding capacity in donors and worse survival of recipients. The CP and MPO levels of organ donors were significantly higher than in healthy controls. The durations of ICU stay increased albumin oxidation. In addition, interleukin-6 (IL-6), IL-8, IL-10, and IL-1ß levels were increased in patients, whereas the interferon-γ (IFN-γ) levels were within the normal range. We conclude that oxidative stress and systemic endotoxemia are present in brain-dead organ donors, which might affect recipient survival. High endotoxin levels might be caused by increased gut permeability and decreased binding capacity of albumin influenced not just by higher albumin oxidation.

Novel immunofluorescence protocol for multimarker assessment of putative disseminating breast cancer stem cells.

PURPOSE: The phenotypical and functional variety of breast cancer cells is well recognized. This variety is evident in primary tumors and in disseminated tumor cells (DTCs) and solid metastases as shown for recognized prognostic factors, such as estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2/neu and also for cancer stem cell markers such as CD44, CD24, or aldehyde dehydrogenase (ALDH). For the development of new therapeutic strategies, the identification and characterization of disseminated breast cancer cells are needed. This requires the use of multiple antibodies (ie, cytokeratin, Her2/neu, ALDH1, CD44, and CD24) labeled with fluorochromes of different colors and spectral image analysis to separate different color spectra. METHODS: We have focused here on putative breast cancer stem cell markers and evaluated the feasibility of triple and quadruple labeling of breast cancer cells. Using breast cancer cell lines we have developed a method optimized for multimarker analysis by employing novel DyLight Technology. Single marker immunofluorescence was performed in 6 replicates, and reproducible results had to be obtained before proceeding to multimarker immunofluorescence. RESULTS: Three of the markers, CD44, ALDH1, and cytokeratin have been directly conjugated with DyLight dyes. CD24 could not be conjugated directly to the fluorescent dye. A labeled secondary antibody was used for visualization. Single and multimarker immunofluorescence gave consistent results throughout the replicates. CONCLUSIONS: This novel protocol will facilitate detection and phenotypical characterization of disseminated tumor cells. In addition, by adding additional markers, distinct subpopulations could be evaluated for the expression of particular therapeutic targets.

Rapid and reliable detection of LINE-1 hypomethylation using high-resolution melting analysis.

OBJECTIVES: The aim of the present study was to evaluate the precision and reproducibility of the LINE-1 high-resolution melting (HRM) assay to detect LINE-1 hypomethylation. DESIGN AND METHODS: We first evaluated a methylated DNA dilution matrix and a panel of human cancer cell lines. We then applied this LINE-1 HRM assay to a set of 37 archival prostate cancer tissue samples. RESULTS: Our LINE-1 HRM assay revealed small and reproducible run-to-run and bisulfite-to-bisulfite variations. As expected, we found a large variation in methylation levels between different cancer cell lines. All results were confirmed with MethyLight and pyrosequencing as indicated by the high correlation coefficient. Finally, we successfully applied the LINE-1 HRM assay to archival prostate cancer tissues. CONCLUSIONS: The present LINE-1 HRM assay represents a novel, accurate, and cost-effective method to measure global hypomethylation, which makes it suitable for high- and low-throughput laboratories.

The role of activation-induced cell death in the higher onset of spontaneous apoptosis of NK cell subsets in patients with metastatic epithelial cancer.

To address the question whether the higher onset of apoptosis of circulating NK cell subsets might be activation induced in cancer patients, surface expression of NKG2D and serum (s) levels of MHC class I chain-related (MIC) proteins in relation to apoptosis marker and CD95 expression on NK cells were evaluated. Patients showed a significantly higher onset of spontaneous apoptosis of CD56dim NK cells. No difference in the CD95 expression could be detected between patients and normal controls (NCs). Patients' CD56bright NK cells demonstrated a higher expression of NKG2D compared to CD56dim NK cells. The sMICB levels showed a higher level in patients versus NCs. No correlation between sMIC protein levels with both NKG2D expression and onset of spontaneous apoptosis of NK cell subsets was found. Our data suggest that the higher onset of apoptosis of circulating NK cell subsets of patients is not triggered by activation-induced cell death.

Optimization of diagnostic ELISA-based tests for the detection of auto-antibodies against tumor antigens in human serum.

Colorectal cancer is one of the most common cancer types worldwide and it continues to be a serious public health problem. Early detection and diagnosis are of great importance in cancer management. At present, diagnostic blood tests are based on the detection of tumor-associated markers such as carcino-embryonic antigen (CEA), the cancer antigen CA19-9 for gastrointestinal cancer, CA15-3 for breast cancer or CA125 for ovarian cancer. The lack of sensitivity and specificity of these markers prevents their general use in cancer screening of an average risk population. Therefore, new cancer biomarkers or better screening methods are necessary to improve the diagnostics of the disease. This study was directed to the optimization of a diagnostic, enzyme linked immunosorbent assay (ELISA) based test to identify and validate new serum markers, such as extracellular Protein Kinase A (ecPKA) and Nicotinamide N-Methyltransferase (NNMT). In this type of assay, the cancer antigens are quantified indirectly - by detecting the presence of auto-antibodies against tumor proteins in human serum. The result of the optimization and validation process was in the case of ecPKA a reproducible and stable assay. In case of NNMT the assay was probably not sensitive enough.

Resistance to apoptosis and expansion of regulatory T cells in relation to the detection of circulating tumor cells in patients with metastatic epithelial cancer.

Regulatory T cells may be crucial in the development of T cell tolerance to malignancies and contribute to immune dysfunctions. We investigated the percentage, activity, and onset of apoptosis of T cell subpopulations by multicolor flow cytometry in metastatic epithelial cancer patients compared to normal controls. Furthermore, a possible relationship between the presence of circulating tumor cells detected by immunocytochemistry and immune cell abnormalities was evaluated. Our study demonstrated a significantly elevated proportion of regulatory T cells in cancer patients (p < 0.001). In contrast to all other T cell subpopulations, regulatory T cells showed comparable Annexin V-binding characteristics in patients and normal controls. No relationship between the detection of circulating tumor cells and immune dysfunction was observed. These results indicate that cancer patients have a higher number of regulatory T cells with resistance to apoptotic stimuli partly responsible for immune dysfunctions as often observed in cancer patients.

Critical evaluation of real-time reverse transcriptase-polymerase chain reaction for the quantitative detection of cytokeratin 20 mRNA in colorectal cancer patients.

We evaluated the usefulness of cytokeratin 20 (CK20) mRNA expression in the quantitative detection of circulating tumor cells in the blood of patients with colorectal cancer (CRC). Blood samples from healthy volunteers (HVs; n = 37), patients with localized (n = 42) and metastatic colorectal cancer (n = 40), and patients with chronic inflammatory bowel disease (CID; n = 15) were examined. After immunomagnetic enrichment using microbeads against human epithelial antigen, total RNA was extracted, reverse transcribed, and analyzed by real-time reverse transcriptase-polymerase chain reaction using the LightCycler instrument. CK20 expression in peripheral blood was found in 46 of 82 (56%) patients with CRC, 8 of 37 (22%) HVs, and 9 of 15 (60%) patients with CID. Levels of CK20 mRNA were significantly higher in blood samples from CRC patients (median 681) than in blood samples from HVs (median 0) (P = 0.001), whereas no difference could be detected between patients with CRC and CID. Although the present technique could not distinguish CRC from CID, the method warrants further efforts to improve sample preparation and tumor cell enrichment, which may render real-time CK20 reverse transcriptase-polymerase chain reaction a feasible technique in identifying circulating tumor cells in peripheral blood of cancer patients.