Diversity of assessing circulating tumor cells (CTCs) emphasizes need for standardization: a CTC Guide to design and report trials.

Hematogenous spreading of tumor cells from primary tumors is a crucial step in the cascade to metastasis, the latter being the most limiting factor for patients' survival prognosis. Therefore, circulating tumor cells (CTCs) have become a field of intensive research. However, the process of isolation and identification of CTCs lacks standardization. This article presents an overview of 71 CTC studies reported in PUBMED since 2000 and focusing on colorectal cancer. These studies are evaluated regarding standardization of CTC isolation and identification, marker proteins used, study population and blood sample quality management, clinical performance, and quality measures. Overall, standardization of CTC assessment seems insufficient. Thus, comparability of CTC studies is hampered and results should be interpreted carefully. We here propose a standardized CTC guideline (CTC Guide) to prospectively design and report studies/trials in a harmonized form. Despite the current interstudy heterogeneity, the data indicate that CTC detection is of clinical relevance and CTCs should be considered as a surrogate prognostic marker. Many studies indicate the high potential for CTCs as prognostic markers, e.g., in colorectal cancer treatment. However, standardized, large-scale multicenter validation studies are still needed to pave the way for clinical implementation of CTC detection that could ameliorate individualized medicine regimes.

A 'waterfall' transfer-based workflow for improved quality of tissue microarray construction and processing in breast cancer research.

A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

Pancreatic carcinoma cell lines reflect frequency and variability of cancer stem cell markers in clinical tissue.

BACKGROUND: Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue. METHODS: Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry. RESULTS: All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity. CONCLUSIONS: Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.

Serum biomarkers for improved diagnostic of pancreatic cancer: a current overview.

PURPOSE: Complete resection constitutes the only curative approach in pancreatic cancer but is possible only in a minority of patients due to advanced stages upon diagnosis. Consequently, early detection is crucial for curative treatment. Clinical routine still lacks efficient, non-invasive screening assays, and 80-90% of pancreatic carcinomas are detected at unresectable stages. A wide range of serum proteins have been in the focus of intensive search for biomarkers specific for pancreatic cancer. This article will give an overview on serum biomarkers with screening potential for pancreatic malignancy. DESIGN AND METHODS: PUBMED database was searched for articles, and 43 manuscripts were selected that provided data regarding biomarkers used, type of assay, study population, sample cohort quality and diagnostic performance. RESULTS: Superior values for diagnostic performance were shown for MIC-1, PAM4, OPN, HSP27, TPS, TSGF, and CAM17.1 as individual markers. Panels of biomarkers comprised CA 19-9, MCSF, CEA, SAA, Haptoglobin, TSGF, CA 242, and HSP27. Individually or in concerted form, sensitivity and specificity ranged from 77 to 100% and 84-100%, respectively. CONCLUSIONS: While the above named markers show high screening potential for pancreatic cancer, standardized validation studies using multiplex assays are required to pave the way for clinical routine application.

The Inventory of Negative Thoughts in Response to Pain: factor structure and psychometric properties in a college sample.

The psychometric properties of the Inventory of Negative Thoughts in Response to Pain (INTRP) were investigated in a sample of undergraduate students. Factor analysis identified three factors: negative self-statement, negative social cognition, and self-blame. Reliabilities of the factor scales were high. No significant gender differences were obtained on the factor scales. Correlations between the factor scales and the nine subscales of the Symptom Checklist 90R were positive and significant, except one. The results support the factor structure and reliability of the INTRP in a sample of college students.

The acute toxicity evaluation of a low-temperature hydraulic fluid.

A low-temperature version of MIL-H-83282 (LT 83282) is a candidate hydraulic fluid to be used as a replacement for the current low-temperature fluid used on Strategic Air Command aircraft. A single neat dose of 0.1 mL LT 83282 into New Zealand White (NZW) rabbit eyes resulted in slight conjunctival irritation for up to 24 hr after treatment in two of nine rabbits. Rinsing the eyes after treatment appeared beneficial. A single treatment of 0.5 mL neat LT 83282 to rabbit skin produced no irritation. A total of 40% of the guinea pigs receiving repeated dermal application of the fluid demonstrated a positive sensitization response. A single oral dose of 5 g LT 83282/kg body weight given to five male and five female Fischer 344 (F-344) rats and a single dermal application of 2 g LT 83282/kg body weight applied to five male and five female NZW rabbits resulted in no deaths. Inhalation exposures to aerosol concentrations of LT 83282 resulted in an LC50 of 2.13 and 1.50 mg/L for male and female F-344 rats, respectively. No clinical signs of acute delayed neurotoxicity were observed in hens twice dosed at limit levels (5 g/kg) and observed for 21 days.

Evaluation of the acute toxicity of silahydrocarbon.

Silahydrocarbon (SHC) is a base stock for a candidate high-temperature hydraulic fluid. Because United States Air Force personnel working with SHC may be exposed to potential health hazards, a complete battery of acute toxicity tests was performed with the fluid. A single neat dose of 0.1 mL SHC into New Zealand White (NZW) rabbit eyes resulted in slight conjunctival irritation 1 hr after treatment in all unflushed eyes and one of three flushed eyes. Conjunctival irritation persisted through 24 hr but dissipated by 48 hr. Rinsing the eyes after treatment was of questionable benefit. A single treatment of 0.5 mL neat SHC to rabbit skin produced negative results for all but a single animal, which had very slight erythema at the test site 48 hr after treatment. Guinea pigs failed to exhibit a sensitization response following repeated application of SHC. A single oral dose of 5 g SHC/kg body weight given to five male and five female F-344 rats and a single dermal dose of 2 g SHC/kg body weight applied to five male and five female NZW rabbits resulted in no deaths or signs of toxic stress. Five male and five female F-344 rats were exposed to 4.8 mg/L (near limit concentration) of aerosolized SHC for 4 hr. All male rats and four female rats survived. During exposure, the animals exhibited signs of eye and upper respiratory irritation.

Repeated-dose gavage studies on polychlorotrifluoroethylene acids.

C8 polychlorotrifluoroethylene (pCTFE) oligomers accumulate preferentially in the liver during long-term oral exposure and appear to be more hepatotoxic than C6 oligomers. A repeated-dose gavage study was initiated to determine the relative contributions of the corresponding C6 (trimer) and C8 (tetramer) acid metabolites to the toxicity of pCTFE in the male Fischer 344 rat. Test animals were dosed once per week for various time periods up to one year. A depression (p less than 0.05) in mean body weight occurred in the highest dose tetramer acid (2.16 mg/kg) group. An increase in hepatic peroxisomal beta-oxidation activity was found in the 2.16 mg pCTFE tetramer acid/kg dose group at the 3-, 6-, and 9-month sacrifice periods. An increase in relative liver weight was seen at all sacrifice periods in this dose group. Hepatocellular cytomegaly was a common finding in the higher dose tetramer acid groups but not in the trimer-treated rat groups.

Effects of a 13-week chloropentafluorobenzene inhalation exposure of Fischer 344 rats and B6C3F1 mice.

Chloropentafluorobenzene (CPFB) has been identified as a candidate simulant for nonpersistent chemical warfare agents. Acute toxicity studies have shown that CPFB has limited adverse effects on laboratory animals. A 21-day inhalation study of rats and mice to 2.5, 0.8, and 0.25 mg CPFB/liter resulted in reduced weight gain in male and female rats exposed at the high concentration only and identified the liver as a potential target organ. This multiconcentration inhalation study was designed to detect a no-observable-effect level associated with repeated exposure to CPFB. Male and female rats and mice were exposed to 250, 50, or 10 mg CPFB/m3 (0.25, 0.05, or 0.01 mg CPFB/liter) for 13 weeks. No treatment-related effects on body weight, clinical chemistries, mortality, absolute or relative organ weight or histopathology were noted.