Cellular Migration Ability Is Modulated by Extracellular Purines in Ovarian Carcinoma SKOV-3 Cells.

Extracellular nucleotides and nucleosides have emerged as important elements regulating tissue homeostasis. Acting through specific receptors, have the ability to control gene expression patterns to direct cellular fate. We observed that SKOV-3 cells express the ectonucleotidases: ectonucleotide pyrophosphatase 1 (ENPP1), ecto-5'-nucleotidase (NT5E), and liver alkaline phosphatase (ALPL). Strikingly, in pulse and chase experiments supplemented with ATP, SKOV-3 cells exhibited low catabolic efficiency in the conversion of ADP into AMP, but they were efficient in converting AMP into adenosine. Since these cells release ATP, we proposed that the conversion of ADP into AMP is a regulatory node associated with the migratory ability and the mesenchymal characteristics shown by SKOV-3 cells under basal conditions. The landscape of gene expression profiles of SKOV-3 cell cultures treated with apyrase or adenosine demonstrated similarities (e.g., decrease FGF16 transcript) and differences (e.g., the negative regulation of Wnt 2, and 10B by adenosine). Thus, in SKOV-3 we analyzed the migratory ability and the expression of epithelium to mesenchymal transition (EMT) markers in response to apyrase. Apyrase-treatment favored the epithelial-like phenotype, as revealed by the re-location of E-cadherin to the cell to cell junctions. Pharmacological approaches strongly suggested that the effect of Apyrase involved the accumulation of extracellular adenosine; this notion was strengthened when the incubation of the SKOV-3 cell with α,ß-methylene ADP (CD73 inhibitor) or adenosine deaminase was sufficient to abolish the effect of apyrase on cell migration. Overall, adenosine signaling is a fine tune mechanism in the control of cell phenotype in cancer. J. Cell. Biochem. 118: 4468-4478, 2017. © 2017 Wiley Periodicals, Inc.

Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT).

The epithelium-mesenchymal transition (EMT) is an important process of cell plasticity, consisting in the loss of epithelial identity and the gain of mesenchymal characteristics through the coordinated activity of a highly regulated informational program. Although it was originally described in the embryonic development, an important body of information supports its role in pathology, mainly in cancerous and fibrotic processes. The purinergic system of inter-cellular communication, mainly based in ATP and adenosine acting throughout their specific receptors, has emerged as a potent regulator of the EMT in several pathological entities. In this context, cellular signaling associated to purines is opening the understanding of a new element in the complex regulatory network of this phenotypical differentiation process. In this review, we have summarized recent information about the role of ATP and adenosine in EMT, as a growing field with high therapeutic potential.

Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata).

Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species.

Vertical jumping biomechanical evaluation through the use of an inertial sensor-based technology.

Progress in micro-electromechanical systems has turned inertial sensor units (IUs) into a suitable tool for vertical jumping evaluation. In total, 9 men and 8 women were recruited for this study. Three types of vertical jumping tests were evaluated in order to determine if the data provided by an IU placed at the lumbar spine could reliably assess jumping biomechanics and to examine the validity of the IU compared with force plate platform recordings. Robust correlation levels of the IU-based jumping biomechanical evaluation with respect to the force plate across the entire analysed jumping battery were found. In this sense, significant and extremely large correlations were found when raw data of both IU and force plate-derived normalised force-time curves were compared. Furthermore, significant and mainly moderate correlation levels were also found between both instruments when isolated resultant forces' peak values of predefined jumping phases of each manoeuvre were analysed. However, Bland and Altman graphical representation demonstrated a systematic error in the distribution of the data points within the mean ±1.96 SD intervals. Using IUs, several biomechanical variables such as the resultant force-time curve patterns of the three different vertical jumps analysed were reliably measured.

The P2RY2 Receptor Induces Carcinoma Cell Migration and EMT Through Cross-Talk With Epidermal Growth Factor Receptor.

Extracellular nucleotides are signaling elements present in the tumor microenvironment; however, their role in tumor growth is not completely understood. In the present study, we asked whether nucleotides regulate cell migration in ovarian carcinoma-derived cells. We observed that 100 µM UTP induced migration in SKOV-3 cells (1.57 ± 0.08 fold over basal), and RT-PCR showed expression of transcripts for the P2RY2 and P2RY4 receptors. Knockdown of P2RY2 expression in SKOV-3 cells (P2RY2-KD) abolished the UTP-induced migration. The mechanism activated by UTP to induce migration involves transactivation of the epidermal growth factor receptor (EGFR) since we observed that the EGFR kinase inhibitor AG1478 and the PI3K inhibitor Wortmannin inhibit this response (to 0.76 ± 0.23 and 0.46 ± 0.14 relative to the control, respectively). In agreement with these observations, UTP was able to modify the phosphorylation state of the EGFR; likewise, the induction of ERK1/2 phosphorylation promoted by UTP was abolished by a 30-60 min treatment with AG1478. Our data also suggested that the enhanced cell migration involves the epithelium to mesenchymal transition (EMT) process, since a 12 h stimulation of SKOV-3 cells with 100 µM UTP showed an increase in vimentin and SNAIL protein levels (459.8 ± 132.4% over basal for SNAIL). Interestingly, treatment with apyrase (10 U/mL) reduces the migration of control cells and induces a considerable enrichment of E-cadherin in the cell-cell contacts, favoring an epithelial phenotype and strongly suggesting that the nucleotides released by tumor cells and acting through the P2RY2 receptor are potential regulators of invasiveness.

Comparative value of microscopy, serology and real time pcr in the diagnosis of asymptomatic canine Leishmania infantum infection

Se investigó la sensibilidad (SE) del examen citológico mediante microscopia óptica (MO) de improntas de bazo y linfonodo, de la prueba de anticuerpos ELISA (inmuno-ensayo ligado a enzima) y de la PCR (reacción en cadena de la polimerasa) a tiempo real (tr), para diagnosticar la infección asintomática por Leishmania infantum en 110 perros aparentemente sanos, del sureste de España. El porcentaje de perros positivos a MO, ELISA y PCRtr fue 2% (2/110), 27% (26/97) y 67% (39/58), respectivamente, aunque el porcentaje de PCR-positivos osciló entre 35-41% para cada tejido individualmente y 9% en sangre. La SE estimada (intervalos de confianza del 95%) de la MO en relación a la PCRtr y al ELISA fue 5% (0-12) y 8% (0-18), respectivamente. Estos resultados confirman que la mayoría de perros aparentemente sanos de una población endémica de L. infantum están infectados, que aproximadamente solo la tercera parte de éstos desarrolla anticuerpos frente al parásito y solo unos pocos tienen suficiente carga parasitaria en tejido linfoide como para ser detectada mediante MO. Consecuentemente, el grado de concordancia de la PCRtr, el ELISA y la MO en el diagnóstico de leishmaniosis canina asintomática es escaso (AU)

The sensitivity (SE) of cytological examination of spleen and lymphnode smears by optical microscopy (OM), antibody-ELISA (enzyme-linked immunosorbent assays) and real-time (rt) PCR (polymerase chain reaction), for diagnosing asymptomatic canine Leishmania infantum infection was investigated in 110 apparently healthy dogs from southeast Spain. The percentage of OM, ELISA and rtPCR positive dogs were 2% (2/110), 27% (26/97) y 67% (39/58), respectively, although the percentage of rtPCR-positive dogs were 35-41% in individual tissues and 9% in blood. The estimated SE (95% confidence interval) of OM relative to the rtPCR and ELISA tests was 5% (0-12) and 8% (0-18), respectively. Results confirm that most apparently healthy dogs from L. infantum endemic areas are infected, that approximately only one third of these infected dogs develop antibodies and that very few have parasite loads that are high enough to allow detection by OM. As a result, the degree of agreement between rtPCR, ELISA and OM for L. infantum diagnosis in subclinnically infected dogs is low (AU)

Frailty assessment based on wavelet analysis during quiet standing balance test.

BACKGROUND: A standard phenotype of frailty was independently associated with an increased risk of adverse outcomes including comorbidity, disability and with increased risks of subsequent falls and fractures. Postural control deficit measurement during quiet standing has been often used to assess balance and fall risk in elderly frail population. Real time human motion tracking is an accurate, inexpensive and portable system to obtain kinematic and kinetic measurements. The aim of this study was to examine orientation and acceleration signals from a tri-axial inertial magnetic sensor during quiet standing balance tests using the wavelet transform in a frail, a prefail and a healthy population. METHODS: Fourteen subjects from a frail population (79±4 years), eighteen subjects from a prefrail population (80±3 years) and twenty four subjects from a healthy population (40±3 years) volunteered to participate in this study. All signals were analyzed using time-frequency information based on wavelet decomposition and principal component analysis. FINDINGS: The absolute sum of the coefficients of the wavelet details corresponding to the high frequencies component of orientation and acceleration signals were associated with frail syndrome. INTERPRETATION: These parameters could be of great interest in clinical settings and improved rehabilitation therapies and in methods for identifying elderly population with frail syndrome.

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain.

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.

Wavelet analysis based on time-frequency information discriminate chronic ankle instability.

BACKGROUND: Ankle sprains are one of the most common lower extremity injuries. Real time human motion tracking is an accurate, inexpensive and portable system to obtain kinematic and kinetic measurements. The purpose of this study was to discriminate between subjects with chronic ankle instability and subjects with stable ankles through inertial tracking technology and force plates. METHODS: Twelve subjects (mean (SD) 23.16 (5.32) years, 174.83 (8.78) cm, 73.58 (17.10) kg) with stable ankles and 13 (mean (SD) 24.69 (5.91) years, 173.31 (9.07) cm, 69.61 (15.32) kg) with chronic ankle instability performed the Star Excursion Balance Test. Time-frequency information based on wavelet decomposition was used for analysing all signals. FINDINGS: Dynamic balance impairment associated with chronic ankle instability was observed in the peak amplitude in the wavelet approximation as well as the absolute sum of the coefficients of the wavelet details of the acceleration, orientation and force signals. These results were found despite Star Excursion Balance Test performance during anterior, posteromedial and posterolateral excursions lead to similar specific reach distances in both limbs in either the chronic ankle instability or stable ankle groups. INTERPRETATION: These parameters could be of great interest in detecting dynamic balance impairment in individuals at risk of sprains that might otherwise go undetected by only reach distance assessment.

[Functional capacity evaluation in a clinical and ambulatory setting: new challenges of accelerometry to assessment balance and muscle power in aging population]. / Valoración de la capacidad funcional en el ámbito domiciliario y en la clínica. Nuevas posibilidades de aplicación de la acelerometría para la valoración de la marcha, equilibrio y potencia muscular en personas mayores.

If we consider a population of free-living individuals, who are 65 years old and even older, a substantial proportion (in the range of 6% to 25%) suffers from many of the elements of the syndrome of frailty. Although the syndrome is complex and still lacks a standard definition, there is a growing consensus about its signs and symptoms. Patients who are afflicted with frailty typically exhibit losses of muscle strength, fatigue easily, are physically inactive, with an increased risk (and fear) of falling, have undergone a recent, unintentional loss of weight, experience impaired cognition and depression, all of which is frequently complicated by a variety of coexistent illnesses. In this context, functional tests to predict disability and frailty are needed. Accelerometry offers a practical and low cost method of objectively monitoring human movements, and has particular applicability to the monitoring of disability in an aging population. Accelerometers have been used to monitor a range of different movements, including gait, sit to stand transfers and postural sway. This review focuses on methodological concepts in the evaluation of skeletal muscle function and monitoring systems (accelerometers and gyroscopes) in each of these areas. An integrated approach is described in which a combination of accelorometry and gyroscopy can be used to monitor a range of different parameters (muscle power, gait and balance) in an aging population in a clinical or out-patient setting.

Valoración de la capacidad funcional en el ámbito domiciliario y en la clínica. Nuevas posibilidades de aplicación de la acelerometría para la valoración de la marcha, equilibrio y potencia muscular en personas mayores / Functional capacity evaluation in a clinical and ambulatory setting: new challenges of accelerometry to assessment balance and muscle power in aging population

Dentro de cualquier población de individuos mayores de 65 años, una proporción sustancial (entre el 6% y el 25%) sufre diferentes síntomas del síndrome de fragilidad. A pesar de la complejidad del terminó fragilidad y de las imprecisiones en cuanto a su definición existe un consenso sobre sus síntomas y signos. Las personas que poseen este síndrome presentan pérdidas de fuerza muscular, fatiga, disminución de la actividad física, con un aumento del riesgo de padecer anorexia-pérdida de peso, delirium, hospitalización, declive funcional, deterioro cognitivo, mortalidad, ingreso en residencias, caídas e inestabilidad. Bajo este contexto, surge la necesidad de desarrollar tests que sean capaces de predecir de la forma más precoz posible la fragilidad y la discapacidad. La acelerometría es una herramienta adecuada para la monitorización de movimientos humanos de una forma objetiva y fiable, aplicable en la vida diaria de los sujetos sin implicar grandes costes. Los acelerómetros están siendo utilizados en la monitorización de diferentes movimientos. Se pueden obtener un amplio abanico de medidas como: clasificación de movimientos, valoración del nivel de actividad física, estimación del gasto de energía metabólica, medida del equilibrio, ritmo de marcha y control al levantarse-sentarse. Combinando la acelerometría con giróscopos y magnetómetros se podrá añadir información relacionada con la orientación y los cambios de posición. Esta revisión analiza las herramientas y tecnologías existentes que puedan llegar a detectar de manera precoz posibles signos y síntomas de la fragilidad y permitan a los individuos vivir autónomamente de forma más prolongada y en condiciones de mayor seguridad (AU)

If we consider a population of free-living individuals, who are 65 years old and even older, a substantial proportion (in the range of 6% to 25%) suffers from many of the elements of the syndrome of frailty. Although the syndrome is complex and still lacks a standard definition, there is a growing consensus about its signs and symptoms. Patients who are afflicted with frailty typically exhibit losses of muscle strength, fatigue easily, are physically inactive, with an increased risk (and fear) of falling, have undergone a recent, unintentional loss of weight, experience impaired cognition and depression, all of which is frequently complicated by a variety of coexistent illnesses. In this context, functional tests to predict disability and frailty are needed. Accelerometry offers a practical and low cost method of objectively monitoring human movements, and has particular applicability to the monitoring of disability in an aging population. Accelerometers have been used to monitor a range of different movements, including gait, sit to stand transfers and postural sway. This review focuses on methodological concepts in the evaluation of skeletal muscle function and monitoring systems (accelerometers and gyroscopes) in each of these areas. An integrated approach is described in which a combination of accelorometry and gyroscopy can be used to monitor a range of different parameters (muscle power, gait and balance) in an aging population in a clinical or out-patient setting (AU)

Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines.

The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.

A membrane associated metalloprotease cleaves Cry3Aa Bacillus thuringiensis toxin reducing pore formation in Colorado potato beetle brush border membrane vesicles.

Insect proteases are implicated in Bacillus thuringiensis insecticidal proteins mode of action determining toxin specificity and sensitivity. Few data are available on the involvement of proteases in the later steps of toxicity such as protease interaction with toxin-receptor complexes and the pore formation process. In this study, a Colorado potato beetle (CPB) midgut membrane metalloprotease was found to be involved in the proteolytic processing of Cry3Aa. Interaction of Cry3Aa with BBMV membrane proteases resulted in a distinct pattern of proteolysis. Cleavage was demonstrated to occur in protease accessible regions of domain III and was specifically inhibited by the metalloprotease inhibitors 1,10-phenanthroline and acetohydroxamic acid. Proteolytic inhibition by a peptide representing a segment of proteolysis in domain III and the metalloprotease inhibitor acetohydroxamic acid correlated with increased pore formation, evidencing that Cry3Aa is a specific target of a CPB membrane metalloprotease that degrades potentially active toxin.

Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say).

Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins. In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.

Simultaneous detection of the immunophenotypic markers and genetic aberrations on routinely processed paraffin sections of lymphoma samples by means of the FICTION technique.

Disciplines such as morphology, immunophenotyping and genetics widely contributed over decades to the understanding of the cellular mechanisms of cancer. To obtain a greater insight into the complex processes of tumorigenesis, scientists have joined their efforts to combine many of the available techniques. Here, we report on the development of a FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms) technique that allows a simultaneous detection of immunophenotypic markers and genetic aberrations on routinely processed lymphoma samples. As the antigen retrieval method seems to play an important role in the final results, we tested the pressure-cooking method at different times (2, 4 and 8 min) using three different buffers (EDTA, Tris-EDTA and citrate), resulting in improved sensitivity for the detection of both immunophenotypic markers and genetic aberrations. We also applied this method to different types of lymphoma using double immunofluorescence assays (including CD30, CD20, CD8 monoclonal antibodies) and several fluorescence in situ hybridization probes to demonstrate that the FICTION technique could be easily applied on paraffin sections in different combinations for the diagnosis and research of cancer.

Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques.

The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found.

Complex cytogenetic abnormalities including telomeric associations and MEN1 mutation in a pediatric ependymoma.

Ependymomas are neuroectodermal tumors of the brain and spinal cord. Some recurrent cytogenetic aberrations have been reported in these tumors, including alterations involving chromosomes 22, 6, and 11. However, consistent molecular alterations have not been identified in ependymal tumors. We studied a recurrent ependymoma in a 3-year-old patient by standard cytogenetic and molecular analysis of TP53 and MEN1 genes. In the present case, we found many of the cytogenetic features previously described as being recurrent in ependymomas, including unstable telomeric alterations. Furthermore, we detected a novel acquired heterozygous mutation in the MEN1 gene. The chromosomal instability produced by the telomeric alterations and the mutation in the MEN1 gene could be important events in the tumorigenesis of ependymomas.

FISHing in the microwave: the easy way to preserve proteins. I. Colocalization of DNA probes and surface antigens in human leukocytes.

Most of the techniques for simultaneous visualization of proteic cell components and DNA probes are difficult to carry out. We have developed an alternative protocol for simultaneous visualization of DNA probes and cell surface antigens in human cells. The method exploits microwave energy to denature DNA and to anneal the probe after antigen fluorescent detection of unfixed cells previously embedded in a microgel. CD3 antigens and whole genome DNA probes or specific repetitive DNA sequences were colocalized in peripheral blood samples. The results show a strong, specific and consistent hybridization pattern in each cell that allowed correlation between cell subtype (PROTEINS) and nuclear phenotype (DNA).

Identification of ins(8;21) with AML1/ETO fusion in acute myelogenous leukemia M2 by molecular cytogenetics.

A high percentage of cases of acute myelogenous leukemia (AML) of the M2 subtype show a rearrangement between the AML1 and ETO genes. The detection of the AML1/ETO fusion has clinical relevance because patients with this subtype have a good prognosis. We present the results of conventional and molecular cytogenetic studies in a patient with acute myelogenous leukemia French-American-British M2 classification, who had a complex karyotype involving chromosomes 8 and 21. Dual-color fluorescence in situ hybridization (FISH) using the AML1/ETO probe demonstrated a recombination of both genes on an add(8) chromosome. The use of other FISH probes (CEP8, c-myc and TEL21) and spectral karyotyping indicated that AML1/ETO fusion occurred as a consequence of a previously undescribed ins(8;21)(q22;q22.1q22.3).