High tumour contamination of leukaphereses in patients with small cell carcinoma of the lung: a comparison of immunocytochemistry and RT-PCR

In small-cell lung carcinoma (SCLC) tumour cell contamination of leukaphereses is unknown. The present study was performed to define appropriate markers for reverse transcriptase polymerase chain reaction (RT-PCR), then to assess the contamination rate of leukaphereses and corresponding bone marrow samples. Immunocytochemistry (ICC) and RT-PCR methods were also compared. Among the 33 patients included, analyses were performed in 16 who had multiple leukaphereses and 17 who had only bone marrow. Leukapheresis products and bone marrow were analysed by ICC using several specific monoclonal antibodies against neural-cell adhesion molecule (N-CAM), epithelial glycoprotein (EGP-40) and cytokeratins (CK). Samples were also analyzed by RT-PCR for expression for N-CAM, synaptophysin, neuron-specific enolase, chromogranin, cytokeratin-18/-19, CEA, EGP-40, apomucin type 1 (MUC-1) and human endothelial cell-specific molecule (ESM-1). Using ICC staining, contaminating tumour cells were detected in 34% of leukaphereses (27% in patients with limited disease and 43% in those with extensive disease). N-CAM was the most reliable marker for detection of contamination. For RT-PCR, CK-19 and CEA were the only appropriate markers. Positive signal rate in leukaphereses increased to 78% (89% for patients with limited disease and 67% for extensive disease). In bone marrow, both techniques were in agreement whereas in leukaphereses, RT-PCR was better than ICC. A high rate of tumour cell contamination was demonstrated not only in bone marrow but also in leukaphereses from SCLC patients. The most appropriate technique was RT-PCR mainly in patients with limited disease. © 2001 Cancer Research Campaign http://www.bjcancer.co

Clinical impact of different detection methods for disseminated tumor cells in bone marrow of patients undergoing surgical resection of colorectal liver metastases: a prospective follow-up study

<p>Abstract</p> <p>Background</p> <p>Large number of patients with colorectal liver metastasis show recurrent disease after curative surgical resection. Identification of these high-risk patients may guide therapeutic strategies. The aim of this study was to evaluate whether the presence of disseminated tumor cells in bone marrow from patients undergoing surgical resection of colorectal liver metastases can predict clinical outcome.</p> <p>Methods</p> <p>Sixty patients with colorectal liver metastases were planned for a curative resection between 2001 and 2007. All patients underwent bone marrow aspiration before surgery. Detection of tumor cells was performed using immunocytochemical staining for cytokeratin (CK-ICC) combined with automated microscopy or indirectly using reverse transcription-polymerase chain reaction (RT-PCR).</p> <p>Results</p> <p>Disseminated tumor cells were found in 15 of the 46 patients (33%) using CK-ICC and in 9 of 44 of the patients (20%) using RT-PCR. Patients with negative results for RT-PCR had a significant better disease-free survival after resection of their liver metastases (p = 0.02). This group also showed significant better overall survival (p = 0.002). CK-ICC did not predict a worse clinical outcome.</p> <p>Conclusions</p> <p>The presence of disseminated tumor cells in bone marrow detected using RT-PCR did predict a worse clinical outcome. The presence of cells detected with CK-ICC did not correlate with poor prognosis.</p

Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays

BACKGROUND: Human adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. METHODS: Water samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization. RESULTS: The analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 10(2) HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. The molecular characterization studies indicated that HAdV-2 was the prevalent serotype. CONCLUSIONS: These results indicate a lack of proper public health measures. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA

Detección y cuantificación del virus de la enfermedad de linfocistis mediante ensayo ICC-RT-PCR (integrated cell culture-RT-PCR)

La enfermedad de linfocistis es la enfermedad de etiología viral más frecuentemente detectada en la acuicultura marina europea, siendo la principal patología de origen vírico descrita en doradas cultivadas. El agente etiológico de esta enfermedad es el virus de la enfermedad de linfocistis (LCDV), miembro del género Lymphocystivirus, perteneciente a la familia Iridoviridae. Se han desarrollado diversos protocolos de PCR y PCR a tiempo real que permiten la detección y cuantificación del LCDV en diversas muestras, si bien no aportan ninguna indicación de la infectividad de los virus detectados. La detección de partículas víricas infectivas requiere la utilización de cultivos celulares, pero en el caso del LCDV la observación de efectos citopáticos (CPE) es difícil y a menudo está sujeta a subjetividad, especialmente en muestras con baja carga vírica. Por este motivo, en el presente trabajo se ha desarrollado un ensayo de ICC-RT-PCR (Integrated Cell Culture-RT-PCR) que permite la detección de partículas infectivas del LCDV. Este ensayo se ha aplicado en combinación con el método del número más probable (NMP) para la determinación del título infectivo en cultivos celulares. El protocolo de ICC-RT-PCR desarrollado permitió la detección de mRNA viral a partir de células SAF-1 inoculadas con un título infectivo de LCDV de 0,1 TCID50/ml, procesadas a los 5 d p.i, mientras que el límite de detección mediante observación de CPE fue de 10 TCID50/ml a 14 d p.i. La sensibilidad de la técnica he permitido la detección de partículas infectivas del LCDV en ejemplares de dorada asintomáticos, donde no se observaron CPE en cultivos celulares inoculados en paralelo y mantenidos hasta 14 d p.i. Este protocolo también se ha aplicado para la determinación del título infectivo de diferentes aislados víricos obtenidos a partir de peces enfermos, reduciéndose de forma considerable el tiempo necesario para realizar la titulación en comparación con el método de la dosis infectiva 50% en cultivos celulares (TCID50) (5 d versus 14-21 d, respectivamente). Así mismo, se han titulado stocks víricos obtenidos en cultivos celulares, donde la carga vírica es inferior al límite de detección del ensayo de observación de CPE. En conclusión, el protocolo de ICC-RT-PCR desarrollado es una técnica sensible, rápida y útil para la detección y cuantificación de LCDV infectivos, lo que la convierte en una herramienta adecuada para el estudio de esta patología viral.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Detekcja rozsianych komórek nowotworowych w szpiku kostnym u pacjentek z rakiem piersi z zastosowaniem nowej metody molekularnej

Objectives: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. Assays based on RT-PCR are available; however, so far they have not been used for routine detection of DTC. Therefore, the purpose of this study was to evaluate a newly established molecular method for the detection of DTC. Materials and methods: BM aspirates from 405 patients were examined. Half of the samples were immediately inserted into ICC and the other half was examined with our newly established molecular method based on RT-PCR. Immunocytochemistry was performed according to the Consensus Recommendations of the German, Austrian, and Swiss Societies of Senology and ISHAGE Working Group (A45B-B3 antibody). RT-PCR was conducted as a one-step real-time assay. Cytokeratin 19-mRNA was amplifi ed. Results: In 142 of 405 (35%) aspirates disseminated tumor cells were detected by RT-PCR. In 34% of patients DTC were detected by ICC. 48% of the BM samples were positive by at least one method. In 73% of the patients identical results were obtained (pCel pracy: Liczne badania pokazały, że obecność rozsianych komórek nowotworowych (RKN) w szpiku kostnym u pacjentek chorych na raka piersi związana jest z niekorzystnym rokowaniem. Metodą standardową w oznaczaniu RKN jest immunocytochemia (ICC). Metody molekularne, takie jak RT-PCR, są również dostępne, dotychczas jednak nie są one stosowane rutynowo w detekcji RKN. Celem pracy była ewaluacja nowej metody molekularnej do oznaczania RKN. Materiał i metody: Zbadane zostały aspiraty szpiku kostnego pochodzące od 405 pacjentek z rakiem piersi. Połowę pobranego szpiku badano immunocytochemicznie, drugą połowę - nową metodą molekularną opartą na technologii RT-PCR. Immunocytochemię przeprowadzano zgodnie z zaleceniami Niemieckiego, Austriackiego i Szwajcarskiego Towarzystwa Chorob Piersi oraz grupy ISHAGE (przeci ciało A45B-B3). RT-PCR przeprowadzano w konwencji One-step, amplifi kacja mRNA cytokeratyny 19 rejestrowana była w czasie rzeczywistym („real-time”). Wyniki: W 142 z 405 (35%) pobranych szpików wykryto rozsiane komórki nowotworowe metodą RT-PCR. U 34% pacjentek wykryto RKN immunocytochemicznie. 48% aspiratów było pozytywnych przy użyciu przynajmniej jednej z metod. W 73% przypadków uzyskano ten sam wynik przy pomocy obu metod (

Detection of micrometastases in peripheral blood of non-small cell lung cancer with a refined immunomagnetic nanoparticle enrichment assay

Fe3O4 particles are currently used as the core of immunomagnetic microspheres in the immunomagnetic enrichment assay of circulating tumor cells (CTCs). It is difficult to further improve the sensitivity of CTC detection or to improve tumor cell-type identification and characterization. In the present study, we prepared immunomagnetic nanoparticles with nanopure iron as the core, coated with anti-cytokeratin 7/8 (CK7/8) monoclonal antibody. These immunomagnetic nanoparticles (IMPs) were used in conjunction with immunocytochemistry (ICC) to establish a refined immunomagnetic nanoparticle enrichment assay for CTC detection in non-small cell lung cancer (NSCLC). The assay was compared with nested reverse transcription polymerase chain reaction (RT-PCR) to detect CK19 mRNA and lung specific X protein (LUNX) mRNA. Human lung adenocarcinoma cell line A549 was used for sensitivity and specificity evaluation. Peripheral blood samples were collected from each group for CTC detection. The average diameter of the immunomagnetic nanoparticles was 51 nm, and the amount of adsorbed antibodies was 111.2 μg/mg. We could detect down to one tumor cell in 5 × 107 peripheral blood mononuclear cells. The sensitivity was consistent with that of nested RT-PCR; however, the false positive rate was significantly reduced. The modified assay combined with ICC did not differ from nested RT-PCR in sensitivity, but it had significantly increased specificity. This approach could, therefore, contribute to identification of micrometastases, re-defining clinical staging, and guiding individual postoperative treatments. The technique shows considerable potential clinical value and further clinical trials are warranted

Expression Level of Kita, Kitb, Kitla, and Kitlb in Zebrafish Gastrointestional Tract

Gastrointestinal (GI) motility is the muscular contractions that move intestinal contents in an anterograde (mouth to anus) direction and is necessary for nutrient absorption and elimination of waste. GI motility is highly coordinated and rhythmic contraction patterns. Interstitial cells of Cajal (ICC), enteric neurons, and smooth muscle cells all regulate GI motility. ICC function as pacemaker cells and determine contraction frequency. ICC growth and development is influenced by Kit, a tyrosine kinase receptor located on the plasma membrane of ICC. TMEM16A is a calcium activated chloride channel which contributes to the slow wave in the GI tract. Constipation, delayed gastric emptying, and bloating have been correlated with deficits of ICC in GI tissues. A functional Kit receptor and stimulation of Kit with Kit ligand is necessary for ICC growth and development. However, little is known about ICC development in adults or in developing GI tissue. The objective for this project is to determine the relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb in the zebrafish model system at several developmental time points. Understanding the temporal and relative expression pattern of these genes is the first step towards a more complete understanding of ICC development and turnover. The zebrafish model system is anatomically similar to the human GI tract and at early time points the zebrafish is transparent. One advantage to this model system is that GI motility may be examined in the intact larvae. RNA was isolated from dissected zebrafish GI tissues and used as template for reverse transcriptase reactions to make eDNA. Relative and temporal expression levels of Kita, Kitb, Kitla, and Kitlb was determined at 5 days post fertilization (dpf), 7 dpf, 11 dpf, 28dpf, and in adult gut tissues using eDNA as template for real time PCR. Kita and Kitla were confirmed as a functional receptor/ligand pair which was first identified in melanocyte migration19. The relative expression data suggests that Kitb and Kitlb are also a functional receptor/ligand pair. Temporal expression data shows high expression of Kitb early in development (5dpf). Besides the early high expression of Kitb, gene expression for all genes of interest peak at 11 dpf. TMEM16A (also called ANOI) was identified as a more accurate marker for gastrointestinal stromal tumors (GIST) than Kit24. RNA isolated from dissected zebrafish GI tract was used to make eDNA which became the template for reverse transcriptase (RT)-PCR and real-tin1e PCR (q-PCR). Anti-ANOI antibodies were used to identify TMEM16A in dissected, fixed zebrafish GI tract. RT-PCR showed that TMEM16A, B, and Care expressed in the zebrafish GI tract. Immunohistochemistry identifies a network of cells in the zebrafish GI tract that is similar in morphology and location to ICC stained by Kit antibodies. Relative and temporal expression was determined using samples isolated at 5, 7, 11, 28dpf, and adult time points. Expression of TMEM16B dominates TMEM16A and B at 28dpf and adult time points