Expression of the filaggrin gene in umbilical cord blood predicts eczema risk in infancy: A birth cohort study.

BACKGROUND: Filaggrin gene (FLG) expression, particularly in the skin, has been linked to the development of the skin barrier and is associated with eczema risk. However, knowledge as to whether FLG expression in umbilical cord blood (UCB) is associated with eczema development and prediction is lacking. OBJECTIVE: This study sought to assess whether FLG expression in UCB associates with and predicts the development of eczema in infancy. METHODS: Infants enrolled in a birth cohort study (n=94) were assessed for eczema at ages 3, 6, and 12 months. Five probes measuring FLG transcripts expression in UCB were available from genomewide gene expression profiling. FLG genetic variants R501X, 2282del4, and S3247X were genotyped. Associations were assessed using Poisson regression with robust variance estimation. Area under the curve (AUC), describing the discriminatory/predictive performance of fitted models, was estimated from logistic regression. RESULTS: Increased level of FLG expression measured by probe A_24_P51322 was associated with reduced risk of eczema during the first year of life (RR=0.60, 95% CI: 0.38-0.95). In contrast, increased level of FLG antisense transcripts measured by probe A_21_P0014075 was associated with increased risk of eczema (RR=2.02, 95% CI: 1.10-3.72). In prediction models including FLG expression, FLG genetic variants, and sex, discrimination between children who will and will not develop eczema at 3 months of age was high (AUC: 0.91, 95% CI: 0.84-0.98). CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated, for the first time, that FLG expression in UCB is associated with eczema development in infancy. Moreover, our analysis provided prediction models that were capable of discriminating, to a great extent, between those who will and will not develop eczema in infancy. Therefore, early identification of infants at increased risk of developing eczema is possible and such high-risk newborns may benefit from early stratification and intervention.

Discovery of serum protein biomarkers in drug-free patients with major depressive disorder.

OBJECTIVE: Major depressive disorder (MDD) is a systemic and multifactorial disorder involving complex interactions between genetic predisposition and disturbances of various molecular pathways. Its underlying molecular pathophysiology remains unclear, and no valid and objective diagnostic tools for the condition are available. METHODS: We performed large-scale proteomic profiling to identify novel peripheral biomarkers implicated in the pathophysiology of MDD in 25 drug-free female MDD patients and 25 healthy controls. First, quantitative serum proteome profiles were obtained and analyzed by liquid chromatography-tandem mass spectrometry using serum samples from 10 MDD patients and 10 healthy controls. Next, candidate biomarker sets, including differentially expressed proteins from the profiling experiment and those identified in the literature, were verified using multiple-reaction monitoring in 25 patients and 25 healthy controls. The final panel of potential biomarkers was selected using multiparametric statistical analysis. RESULTS: We identified a serum biomarker panel consisting of six proteins: apolipoprotein D, apolipoprotein B, vitamin D-binding protein, ceruloplasmin, hornerin, and profilin 1, which could be used to distinguish MDD patients from controls with 68% diagnostic accuracy. Our results suggest that modulation of the immune and inflammatory systems and lipid metabolism are involved in the pathophysiology of MDD. CONCLUSIONS: Our findings of functional proteomic changes in the peripheral blood of patients with MDD further clarify the molecular biological pathway underlying depression. Further studies using larger, independent cohorts are needed to verify the role of these candidate biomarkers for the diagnosis of MDD.

The use of citrullinated peptides for the diagnosis and prognosis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes joint inflammation and extra-articular manifestations. To prevent progressive and irreversible structural damage, early diagnosis of RA is of paramount importance. Antibodies directed against citrullinated proteins and peptides (ACPAs) are the most specific serological markers available for diagnosing RA. ACPAs may be detected several years before symptoms appear, and their presence at disease onset is a good predictor of the development of erosive joint lesions. Synthetic peptides can replace cognate proteins in solid-phase assays for specific autoantibody recognition in RA patients. The use of synthetic peptides instead of proteins represents an advantage in terms of the reproducibility of such immunoassays. They give absolute control over the exact epitopes presented. Furthermore, it is difficult to prepare sufficient amounts of high-quality antigenic proteins with a well-defined degree of citrullination. Synthetic citrullinated peptides, in contrast, are easily obtained in a pure form with a well-defined chemical structure and the epitopes can be precisely oriented in the plate by covalent binding of the peptides. We have recently obtained and highlighted the application of chimeric peptides bearing different citrullinated protein domains for the design of RA diagnosis systems. Our results indicate that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported (fibrin, vimentin and filaggrin) helps to identify a particular subset of RA patients.

Association between P478S polymorphism of the filaggrin gene & atopic dermatitis.

BACKGROUND & OBJECTIVES: Atopic diseases, including atopic dermatitis (AD), allergy and asthma, are complex diseases resulting from the effect of multiple genetic and interacting environmental factors on their pathophysiology. The genetic basis is incompletely understood; however, recent studies have shown an association between loss-of-function variants of the filaggrin gene (FLG) and atopic dermatitis. The aim of this study was to determine whether FLG variants can serve as a predictor for atopic diseases in Korean individuals. METHODS: A total of 648 subjects were genotyped for the FLG P478S (rs11584340, C/T base change) polymorphism (322 patients and 326 controls). Serum levels of free fatty acids (FFA) and IgE were later stratified to determine the effects of the FLG polymorphism on AD. RESULTS: A significant difference in genotype frequency was found between AD patients and controls in the FLG P478S polymorphism. The FLG P478S T allele carrier (TT+TC) was associated with AD risk (odds ratio = 1.877, 95% confidence interval 1.089 to 3.234). In addition, the P478S T allele was related to high levels of FFA in AD patients (471.79 ± 298.96 vs. 333.54 ± 175.82 µg eq/l, P <0.05). INTERPRETATION & CONCLUSIONS: The results of the present study suggest that the FLG P478S polymorphism alone and combined with other factors influences FFA levels and increases the susceptibility to AD.

The pathological characteristics of glioma stem cell niches.

Brain tumor stem cells (BTSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. Recent studies suggest that stem cells are controlled by a particular microenvironment known as a "niche". We therefore analysed human glioma tissues and found that the CD133(+) and nestin(+) niches are perivascularly localized in all glioma tissues. Furthermore, there is a positive correlation between the CD133(+) niches and CD133(+) blood vessels, which is similar to the correlation between the nestin(+) niches and nestin(+) blood vessels. We demonstrate that both CD133(+) blood vessels and nestin(+) blood vessels have an important role in maintaining the structure of the glioma stem cell niche. Moreover, the abundance of CD133(+) niches and nestin(+) niches increases significantly as tumor grade increases. These findings provide a new insight into the biology of BTSC and open a new perspective for targeted therapy against the brain tumors.

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord.

BACKGROUND: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). RESULTS: Here we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. CONCLUSION: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Erythropoietin protects against apoptosis and increases expression of non-neuronal cell markers in the hypoxia-injured developing brain.

Erythropoietin (EPO) is a cytokine hormone with cytoprotective effects in many tissues including the brain. Although the benefits of administration of recombinant human EPO (rhEPO) for neonatal hypoxic brain injury have been demonstrated in neuronal tissue, the effect on non-neuronal cell populations is unclear. We tested the hypothesis that rhEPO would not only protect neuronal cells but also glial cells at a stage of brain development where their maturation was particularly sensitive, and also protect the vasculature. This was evaluated in a rat model of hypoxic injury. 1000 IU/kg rhEPO was delivered intraperitoneally at the start of 4 h hypoxia or normoxia. Treatment groups of neonatal rats (day of birth, at least N = 10 per group) were as follows: normoxia; normoxia plus rhEPO; hypoxia (8% FiO(2) delivered in temperature-controlled chambers); and hypoxia plus rhEPO. Day of birth in rats is equivalent to human gestation of 28-32 weeks. The effects of rhEPO administration, especially to non-neuronal cell populations, and the associated molecular pathways, were investigated. Apoptosis was increased with hypoxia and this was significantly reduced with rhEPO (p < 0.05). The neuronal marker, microtubule-associated protein-2, increased in expression (p < 0.05) when apoptosis was significantly reduced by rhEPO. In addition, compared with hypoxia alone, rhEPO-treated hypoxia had the following significant protein expression increases (p < 0.05): the intermediate filament structural protein nestin; myelin basic protein (oligodendrocytes); and glial fibrillary acidic protein (astrocytes). In conclusion, rhEPO protects the developing brain via anti-apoptotic mechanisms and promotes the health of non-neuronal as well as neuronal cell populations at a time when loss of these cells would have long-lasting effects on brain function.

Filaggrin null mutations and association with contact allergy and allergic contact dermatitis: results from a tertiary dermatology clinic.

BACKGROUND: Filaggrin null (FLG) mutations lead to skin barrier disruption with a reduced resistance towards exogenous agents and also influence the course of disease in atopic dermatitis. OBJECTIVES: To examine the association between FLG mutations and contact allergy, polysensitization, hand eczema at first appearance of disease, occurrence, and course of dermatitis. METHODS: A venous blood sample from 430 individuals was genotyped for FLG mutations R501X and 2282del4 with polymerase chain reaction followed by typing through hybridization to paramagnetic polystyrene beads and analysis on a BioPlex 200. All individuals had a minimum of one positive patch test reaction. RESULTS: In all, 3.5% were 2282del4 heterozygote and 5.1% were R501X heterozygote. An odds ratio (OR) of 1.49 [95% confidence interval (CI) 0.74-3.00] was found for nickel allergy, OR 0.84 (95% CI 0.41-1.74) for polysensitization, OR 0.78 (95% CI 0.25-2.43) for dermatitis, OR 0.96 (95% CI 0.48-1.92) for hand eczema at debut, OR 1.25 (95% CI 0.99-1.57) for duration of disease, and OR 0.76 (95% CI 0.59-0.97) for age at onset. CONCLUSIONS: No association between nickel allergy, polysensitization, hand eczema at first appearance or occurrence of dermatitis, and FLG mutations was found. However, patients with FLG mutations had an earlier age of onset compared with the wild-type genotype and a trend towards longer duration of disease.

Expression of the stem cell marker nestin in peripheral blood of patients with melanoma.

BACKGROUND: There is continued interest in markers indicative of circulating melanoma cells. Nestin is a neuroepithelial intermediate filament protein that was found to be expressed in melanoma and in various cancer stem cells. OBJECTIVES: We investigated expression of nestin in peripheral blood of patients with melanoma. METHODS: We analysed nestin expression by flow cytometry and by quantitative reverse transcription-polymerase chain reaction both in tissues (n = 23) and in blood samples (n = 102) from patients with American Joint Committee on Cancer stage III-IV melanoma. Forty-six negative controls were also added. RESULTS: Flow cytometry did not reveal nestin-expressing cells in peripheral blood of healthy volunteers. In patients with melanoma, however, nestin protein was expressed in a proportion of melanoma cells enriched from peripheral blood by immunomagnetic sorting. In melanoma tissue samples a significant correlation was found between mRNAs coding for nestin and tyrosinase (P = 0.001) and melan-A (P = 0.002), whereas in blood a significant correlation was observed only for tyrosinase (P = 0.015), but not for melan-A (P = 0.53). Nestin expression was higher in stage IV patients compared with stage III/IV with no evidence of disease, in patients with high tumour burden, and was positively correlated to expression of tyrosinase and melan-A. CONCLUSIONS: Nestin was found to be an additional marker of interest for circulating melanoma cells. Prospective studies should investigate its potential added informative value in comparison with markers already in use for melanoma cell detection.

Peripherin-IgG association with neurologic and endocrine autoimmunity.

Peripherin-IgG has been reported a pertinent autoantibody in non-obese type 1 diabetic (NOD) mice. However, it has not previously been recognized in any human disease. In blinded evaluation of serum for markers of neurological autoimmunity in a high-volume diagnostic laboratory, we incidentally identified 26 patients (61% female) with an IgG that bound selectively to neural elements in enteric ganglia, sympathetic nerve trunks and discrete nerve tracts in mid-brain and hind-brain. The target antigen was identified as peripherin, a 55kDa - type III intermediate filament protein. Review of clinical histories revealed that 54% of seropositive patients had dysautonomia (predominantly gastrointestinal dysmotility), 30% had neuropathies with varied sensory symptoms and 35% had clinical or serological evidence of endocrinopathy (type 1 diabetes, thyroiditis or premature ovarian failure). Collectively, 73% had autonomic dysfunction or endocrinopathy. None of 173 healthy subjects was seropositive. Subsequent western blot evaluation of archival sera from patients with small fiber/autonomic neuropathies (with or without endocrinopathy) revealed a 33% seropositivity rate for peripherin-IgG. Our further demonstration that peripherin-immunoreactive autonomic fibers in pancreas, thyroid and ovary are juxtaposed to endocrine epithelium, complement our clinical observations in suggesting that neuronal elements may be a pertinent initial target for immune attack in multiple forms of endocrine autoimmunity (intermolecular epitope spreading). It remains to be determined whether or not peripherin-IgG is predictive for development of small fiber neuropathy (autonomic or somatic).

A comparison of performance of anti-cyclic citrullinated peptide 2 and citrullinated protein antibodies in the diagnosis of rheumatoid arthritis in Iranian patients.

Antibodies to citrullinated proteins and rheumatoid factor (RF) are widely used in patients with rheumatoid arthritis (RA) and the antibodies to citrullinated proteins appear to be the most specific markers of the disease. The objective was to compare the diagnostic performance of the anti-cyclic citrullinated peptide 2 (anti-CCP2) and citrullinated protein Antibodies (CPA) with RF in the diagnosis of RA. Serum samples of 139 patients with RA and 131 patients with other rheumatic diseases were checked for anti-CCP2, CPA uses citrullinated recombinant rat filaggrin as the antigen assay, and RF. The specificity, sensitivity, and receiver operating characteristic (ROC) of tests were then compared. The sensitivity of anti-CCP2, CPA, and RF were 82.7, 83.5, and 61.5%, respectively. The specificities of the tests were 91.2, 78.6, and 90.5%, respectively. The area under ROC curves for the tests were 0.925, 0.890, and 0.847, respectively. Exclusion of overlaps was associated with improved specificity for CPA but no change in the specificity of RF and anti-CCP. The sensitivity of anti-CCP2, CPA, and RF were 66.7, 77.8, and 51.9% for patients with early RA, respectively. The findings of the present study indicate that anti-CCP2 might be of a better diagnostic value for the diagnosis of RA. They also showed that CPA and in the second place anti-CCP2 were useful in the diagnosis of early RA.

Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis.

BACKGROUND: Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination. OBJECTIVE: We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients. METHODS: We examined 24 Japanese AD patients with "FLG-shotgun" method. RESULTS: Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients. CONCLUSION: We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.

[Clinical research on angiogenesis in colorectal carcinoma and expression of CK20 mRNA in peripheral blood].

OBJECTIVE: To investigate the correlation between microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in tissues of colorectal carcinoma and the micrometastasis of tumor cells in these patients' peripheral blood. METHODS: The MVD and expression of VEGF were evaluated immunohistochemically while the micrometastasis of tumor cells in these patients' peripheral blood was detected by RT-PCR method. RESULTS: The average count of MVD in high and middle differentiation grade was 30.2 +/- 12.7, while in low differentiation grade 86.6 +/- 19.1. The expression of VEGF was positive in 26 patients (44.8%). The MVD and positive expression of VEGF were correlated to differentiations. stage and metastasis of colorectal carcinoma. CK(20) mRNA was found in peripheral blood of 32 patients (55.2%) and the positive rate was up to 60.4% 48 hours after operation, among which positive rate in the radical resection group was 47.7% and in the non-radical resection group 85.7%. 11 out of 21 patients positive in CK(20) mRNA turned to negative 7-14 d after radical resection, while 11 out of 12 patients remained positive at the same time after non-radical resection. The expression of CK(20) mRNA was correlated to the stage and metastasis of the cancer. The MVD and positive expression of VEGF were higher in patients with positive expression of CK(20) mRNA. CONCLUSIONS: The MVD and positive expression of VEGF were correlated to differentiation, stage and metastasis of colorectal carcinoma. The angiogenesis in tissues of colorectal carcinoma was closely related to the micrometastasis of tumor cells in these patients' peripheral blood. The detecting of CK(20)mRNA by RT-PCR may be a sensitive method for evaluating the micrometastasis colorectal carcinoma in peripheral blood and help in prognosis prediction, effect assessment and guidance of multipurpose therapy.

Human antibody SC-1 reduces disseminated tumor cells in nude mice with human gastric cancer.

Advanced gastric cancer is a systemic disease that requires adjuvant therapy targeted at eliminating disseminated tumor cells (DTCs). We investigated whether the apoptosis-inducing human monoclonal IgM antibody SC-1 was able to reduce the number of disseminated gastric cancer cells in blood and bone marrow. Human gastric tumor specimens with positive expression of the SC-1 receptor were transplanted in nude mice with metastasizing gastric cancer. After tumor growth (4-6 weeks) animals were randomly allocated to intraperitoneal 100 microg SC-1 (n=23) or 100 microg human IgM (n=23). One week later, animals were sacrificed and blood and bone marrow specimens were obtained. A nested RT-PCR for cytokeratin 20 (CK-20) from blood and bone marrow of mice was performed for detection of disseminated tumor cells. Animals receiving SC-1 had significantly fewer DTCs than did control animals (p=0.0011). None of the SC-1 mice had DTCs simultaneously in both blood and bone marrow versus four of the control animals (p=0.0363). The reduction of DTCs in SC-1 animals was due to reduction in bone marrow (p=0.032 compared to controls), but not in blood (p=0.1158). Treatment with SC-1 significantly reduced the number of DTCs in bone marrow in this animal model.

Detection and quantification of circulating tumor cells in patients with esophageal cancer by real-time polymerase chain reaction.

Polymerase chain reaction (PCR) amplification was used for detecting circulating tumor cells. However, PCR was so sensitive that it detected a very low level of mRNA with no relevance to tumor cells. We analyzed the degree of micro-tumor spread in esophageal cancer patients using quantitative PCR. Samples were collected from 28 patients and 35 controls. Real-time quantitative PCR (LightCycler) was employed for the detection of carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20). In the CEA and CK-20 mRNA assays, 7 and 3 out of 28 patients, respectively, showed higher mRNA levels in peripheral blood than the normal range based on values of controls (mean+/-2SD). Eleven out of 19, 4 out of 14, and 2 out of 5 patients showed higher CEA mRNA levels in the samples from tumor drainage vein, costal bone marrow, and thoracic duct lymph, respectively. One of the 7 patients who showed higher CEA mRNA levels in pretreatment peripheral blood is currently free from disease. These findings reveal that quantitative PCR can discriminate high levels of cancer-specific expression from low levels of illegitimate expression in blood. They also suggest that the identification of circulating tumor cells by the CEA mRNA assay is a reliable means of predicting early recurrence.

[Significance of CK20 mRNA expression in peripheral blood of colorectal cancer patients by real-time fluorescent quantitative RT-PCR].

OBJECTIVE: To detect the expression of cytokeratin 20 (CK20) mRNA in peripheral blood of colorectal carcinoma and to discuss its clinical value. METHODS: Real-time fluorescent quantitative RT-PCR was used to detect the CK20 mRNA expression in the peripheral blood of 51 patients with colorectal carcinoma and 30 healthy volunteers. RESULTS: 27.45% of the patients showed CK20 mRNA expression, while it was 6.67% for the control group (P<0.025). With the progress of Dukes' stages, the expression level of CK20 mRNA increased, but there was no statistic significance (P<0.05). More samples in Dukes'C and D than in Dukes'A and B stages showed >10 copies/ml. CONCLUSION: The detection of CK20 mRNA expression in peripheral blood of patients with colorectal carcinoma may be helpful to identify early shedding tumor cells. It is also useful to monitor the progression of the disease and observe the effect of clinical treatment.

Hematogenous tumor cell dissemination during colonoscopy for colorectal cancer.

BACKGROUND: It has long been suspected that mechanical influences may enhance the release of viable colorectal cancer cells into the circulation. The objective of this study was to determine the extent of hematogenous tumor cell spread in colorectal cancer patients during colonoscopy. METHODS: Peripheral venous blood samples were taken before and after colonoscopy from 44 patients with colorectal cancer. Blood samples were examined using a reverse-transcriptase polymerase chain reaction assay to amplify cytokeratin 20 transcripts. RESULTS: Eleven patients with colorectal cancer displayed circulating tumor cells before and after colonoscopy (25%), whereas tumor cells were detected in six of 44 patients (14%) only after the procedure (p = 0.03, McNemar's test: tumor cell detection before after colonoscopy). All control samples consistently tested negative. CONCLUSIONS: Mechanical forces may result in enhanced release of viable colorectal cancer cells into the circulation; however, the clinical significance of these results needs to be clarified.

Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics.

BACKGROUND: Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection. METHODS: We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma. RESULTS: We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 microm filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent. CONCLUSIONS: Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis.

Detection of circulating tumor cells by cytokeratin 20 and prostate stem cell antigen RT-PCR in blood of patients with gastrointestinal cancers.

BACKGROUND: Detection of circulating tumor cells in blood may be an important diagnostic and prognostic factor in the management of tumor patients. The present study aimed to examine whether cytokeratin 20 (CK-20) and prostate stem cell antigen (PSCA) are useful markers for the detection of disseminated cancer cells in the blood of tumor patients. MATERIALS AND METHODS: A nested RT-PCR assay was used to detect CK-20 and PSCA mRNA in blood samples from 18 healthy donors, 15 patients with non-malignant disease, 9 patients with benign tumors and 47 patients with malignant tumors (11 pancreatic carcinoma, 8 gastric cancer, 15 colorectal carcinoma and 13 miscellaneous tumors). RESULTS: CK-20 expression was observed in the peripheral blood of 19 out of 47 (40.4%) patients with malignant tumors, 2 out of 9 (22.2%) patients with benign tumors and 3 out of 15 (20%) patients with non-tumor diseases. PSCA expression was present in the blood of 22 out of 47 (46.8%) patients with malignant tumors and particularly in 7 out of 11 (63.6%) patients with pancreatic cancer. CK-20 and PSCA expression was not observed in blood samples from healthy donors. There was a relationship between PSCA expression and tumor stage. CONCLUSION: The present results demonstrate that it is possible to apply a simple and reliable method for the detection of circulating tumor cells based on CK-20 and PSCA RT-PCR assays.

Detection of disseminated tumour cells in blood and bone marrow samples of patients undergoing hepatic resection for metastasis of colorectal cancer.

BACKGROUND: In 50-60 per cent of patients who undergo hepatic resection for metastasis of colorectal cancer the first site of tumour recurrence is extrahepatic, indicating the presence of more extensive disease at the time of resection. The aim of this study was to evaluate whether the presence of disseminated tumour cells in blood and bone marrow could predict extrahepatic tumour recurrence. METHODS: Cytokeratin 20 (CK20) reverse transcriptase-polymerase chain reaction was used to study the presence of tumour cells in preoperative peripheral blood and bone marrow samples from 41 patients with liver metastasis scheduled for surgical resection. RESULTS: CK20 expression was detected in six of 41 peripheral blood samples and in eight of 32 bone marrow samples. There was no correlation between CK20-positive samples and subsequent extrahepatic recurrence. Positive blood samples did, however, correlate with high serum carcinoembryonic antigen level and large tumour volume. None of the 14 patients previously treated with chemotherapy had CK20-positive samples, whereas six of 27 blood and eight of 20 bone marrow samples were positive in the chemotherapy-naive group. CONCLUSION: Although the number of patients in this study is limited, the presence of disseminated tumour cells did not predict subsequent extrahepatic recurrence. The results strongly suggest that the presence of circulating tumour cells in peripheral blood may reflect transient shedding of tumour cells related to large tumour volume.