Somatic variation in normal tissues: friend or foe of cancer early detection?

BACKGROUND: Seemingly normal tissues progressively become populated by mutant clones over time. Most of these clones bear mutations in well-known cancer genes but only rarely do they transform into cancer. This poses questions on what triggers cancer initiation and what implications somatic variation has for cancer early detection. DESIGN: We analyzed recent mutational screens of healthy and cancer-free diseased tissues to compare somatic drivers and the causes of somatic variation across tissues. We then reviewed the mechanisms of clonal expansion and their relationships with age and diseases other than cancer. We finally discussed the relevance of somatic variation for cancer initiation and how it can help or hinder cancer detection and prevention. RESULTS: The extent of somatic variation is highly variable across tissues and depends on intrinsic features, such as tissue architecture and turnover, as well as the exposure to endogenous and exogenous insults. Most somatic mutations driving clonal expansion are tissue-specific and inactivate tumor suppressor genes involved in chromatin modification and cell growth signaling. Some of these genes are more frequently mutated in normal tissues than cancer, indicating a context-dependent cancer-promoting or -protective role. Mutant clones can persist over a long time or disappear rapidly, suggesting that their fitness depends on the dynamic equilibrium with the environment. The disruption of this equilibrium is likely responsible for their transformation into malignant clones and knowing what triggers this process is key for cancer prevention and early detection. Somatic variation should be considered in liquid biopsy, where it may contribute cancer-independent mutations, and in the identification of cancer drivers, since not all mutated genes favoring clonal expansion also drive tumorigenesis. CONCLUSION: Somatic variation and the factors governing homeostasis of normal tissues should be taken into account when devising strategies for cancer prevention and early detection.

Genomewide miRNA profiling of oral lichenoid disorders and oral squamous cell carcinoma.

OBJECTIVE: To dissect the aberrant microRNA profile of oral lichenoid disorders (OLD) by analyzing the larger set of OLD samples tested so far. MATERIALS AND METHODS: MicroRNA expression profiles were assessed using TLDA card in 32 samples (16 OLD, 8 OSCC, and 8 control). The findings were validated using RT-qPCR in an independent cohort of 91 samples. RESULTS: We identified 20 differentially expressed microRNAs in OLD, of which several are functionally related to cell proliferation, response to organic substances, or immune processes. Further validation of the top-ranked microRNAs revealed that they were all aberrantly expressed in OLD. CONCLUSION: We have identified a new microRNA signature associated with OLD that may provide a meaningful basis for better understanding the physiopathology of the disease. In addition, we validated seven microRNAs whose expression was shown to be higher in OLD tissue in comparison with the control and OSCC tissues.

Epitelización inducida por células troncales derivadas del tejido adiposo / Induced epithelialization by adipose tissue derived stem cells

El tratamiento de lesiones con pérdida de tejido cutáneo ha mejorado notablemente con el advenimiento de la bioingeniería tisular. Una alternativa en desarrollo es la utilización de sustitutos dérmicos combinados con células troncales derivadas del tejido adiposo autólogo. Estudios previos nos muestran que con esta técnica es posible optimizar la angiogénesis y la síntesis de colágeno, sin embargo potenciar la epitelización es un tema pendiente por resolver. En el presente estudio evaluamos la progresión y diferenciación epitelial en un período de tiempo prologando. Obtuvimos las células troncales a partir del tejido adiposo (ASC) de la región inguinal de 4 ratas Sprague Dawley. Cultivamos las células frescas en una matriz de Integra(R) durante un período total de 48 horas, y las marcamos con un vector lentiviral-GFP (proteína fluorescente verde). Posteriormente, injertamos en las mismas ratas la matriz dérmica con células troncales y un implante contralateral sin células, como control. A las 4 semanas, evaluamos el avance epitelial mediante planimetría de superficie e histología. Los resultados macroscópicos muestran que el cierre de la herida por contracción de los bordes no tiene diferencias significativas (82,63% ± 3,4% vs. 80,66% ± 3,89%; p = 0,08), pero el cierre por epitelización fue significativamente mayor en el lado intervenido con ASCs (93,47% ± 5,98% vs. 79,88% ± 6,28%; p = 0,0028). Todas las muestras obtuvieron tinción positiva para el anticuerpo anti-citoqueratina 34²E12 y el avance epitelial lineal cuantificado por microscopía resultó significativamente mayor en el lado con ASCs (6408 ± 275¼m vs. 5375 ± 250¼m; p < 0,001). Identificamos las células GFP positivas formando parte de la dermis regenerada, no así en la epidermis. En conclusión, las células troncales derivadas del tejido adiposo autólogo sembradas en una matriz de Integra(R) aumentan la formación epitelial significativamente, probablemente por un mecanismo de inducción más que de diferenciación (AU)

The treatment of injuries with loss of skin tissue has improved significantly with the advent of bioengineered tissue. Previous studies showed that the use of dermal substitutes combined with autologous Adipose-derived Stem Cells (ASCs) improve angiogenesis and collagen synthesis; however, epithelialization is an outstanding issue to be resolved. In the present study, we evaluated the epithelial progression and differentiation in an extended period of time. We obtained the adipose tissue derived stem cells (ASCs) from the inguinal fat pad of 4 Sprague Dawley rats. The non-expanded cells were cultivated in a dermal scaffold (Integra® ) for 48 hours and marked with a green fluorescent protein (GFP) lentiviral vector. The scaffold plus stem cells and a contralateral cell-free scaffold (control) were implanted in the same rats. After 4 weeks, epithelial surface was assesses by planimetry and histology. The results show that macroscopic wound closure by contraction from the edges has no significant differences (82.63% ± 3.4% vs. 80.66% ± 3.89%, p = 0.08), but the closure by epithelialization was significantly higher in the side with stem cells (93.47% ± 5.98% vs. 79.88% ± 6.28%, p = 0.0028). All samples were positive for staining with anti-cytokeratin antibody 34²E12 and linear epithelial advancement quantified by microscopy was significantly higher in the side with stem cells (6408 ± 275¼m vs. 5375 ± 250¼m, p < 0.001). GFP positive cells were identified as part of the regenerated dermis but not the epidermis. In conclusion, the autologous adipose tissue derived stem cells seeded in a Integra® scaffold significantly increase epithelial formation, most likely by an induction mechanism rather than affecting differentiation (AU)

Global DNA methylation: uncommon event in oral lichenoid disease.

OBJECTIVES: Accumulating evidence indicates that aberrant DNA methylation is closely related to oral carcinogenesis, and it has been shown that methylation changes might be used as prognostic biomarker in oral squamous cell carcinoma. Oral lichenoid disease (OLD) is the most common oral potentially malignant disorder in our region. The aim of this study was to perform the first wide DNA methylation study in OLD in order to investigate the relevance of DNA methylation changes in this premalignant disorder. MATERIALS AND METHODS: Two different Illumina microarray platforms, namely the GoldenGate Cancer Panel I and the HumanMethylation27 DNA Analysis BeadChip, were utilized in the discovery phase to interrogate the methylation profile of 59 OLD cases and 9 healthy individuals. Top-ranked genes were further validated by pyrosequencing in a second sample set consisting of 160 OLD and 65 controls. RESULTS: Our results show that the frequency of aberrant DNA methylation is rare in OLD, and this finding was further corroborated by pyrosequencing in the biological validation. CONCLUSIONS: These findings reinforce the notion that molecular alterations associated with oral carcinogenesis do not seem to be common events in OLD, which in turn might explain the low rate of malignization of this disorder.

A microRNA-based prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material.

BACKGROUND: Diagnosis is jeopardised when limited biopsy material is available or histological quality compromised. Here we developed and validated a prediction algorithm based on microRNA (miRNA) expression that can assist clinical diagnosis of lung cancer in minimal biopsy material to improve clinical management. METHODS: Discovery utilised Taqman Low Density Arrays (754 miRNAs) in 20 non-small cell lung cancer (NSCLC) tumour/normal pairs. In an independent set of 40 NSCLC patients, 28 miRNA targets were validated using qRT-PCR. A prediction algorithm based on eight miRNA targets was validated blindly in a third independent set of 47 NSCLC patients. The panel was also tested in formalin-fixed paraffin-embedded (FFPE) specimens from 20 NSCLC patients. The genomic methylation status of highly deregulated miRNAs was investigated by pyrosequencing. RESULTS: In the final, frozen validation set the panel had very high sensitivity (97.5%), specificity (96.3%) and ROC-AUC (0.99, P=10(-15)). The panel provided 100% sensitivity and 95% specificity in FFPE tissue (ROC-AUC=0.97 (P=10(-6))). DNA methylation abnormalities contribute little to the deregulation of the miRNAs tested. CONCLUSION: The developed prediction algorithm is a valuable potential biomarker for assisting lung cancer diagnosis in minimal biopsy material. A prospective validation is required to measure the enhancement of diagnostic accuracy of our current clinical practice.

Cytometric analysis of oral scrapings of patients with oral lichen planus.

OBJECTIVE: Aneuploidy has been associated with malignant and premalignant oral lesions. In the past few years, its application in oral precancerous lesions and its prognostic meaning have been controversial issues. The aim of our study was to characterize alterations in DNA content by automated DNA image cytometry in oral scrapings of patients with oral lichen planus. METHODS: Cytological samples from 40 patients clinicopathologically diagnosed with oral lichen planus were analysed by DNA image cytometry. RESULTS: All the cases were classified as diploid, showing a predominant population of cells with normal DNA content (DNA index, 0.85-1.15). Atrophic/erosive lesions showed a higher percentage of tetraploid cells when compared with reticular/papular lesions but this was not statistically significant (P = 0.09). CONCLUSIONS: Aneuploidy does not seem a common event in oral lichen planus lesions. However, we consider that the use of DNA image cytometry of oral scrapings may be an easy and helpful methodology in the follow-up of patients with these lesions.

Orchidectomy increases the formation of non-endothelial thromboxane A2 and modulates its role in the electrical field stimulation-induced response in rat mesenteric artery.

The aim of this study was to analyze whether endogenous male sex hormones influence the release of thromboxane A2(TXA2) and its role in the electrical field stimulation (EFS)-induced response, as well as the mechanism involved. For this purpose, endothelium-denuded mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used to measure TXA2 release; EFS-induced response, nitric oxide (NO), norepinephrine (NA), and prostaglandin (PG) I2 release were also measured in the presence of the TXA2 synthesis inhibitor furegrelate. Orchidectomy increased basal and EFS-induced TXA2 release. Furegrelate decreased the EFS-induced contraction in arteries from control rats, but did not modify it in arteries from orchidectomized rats. The EFS-induced neuronal NO release and vasodilator response were increased by furegrelate in arteries from control rats, but were not modified in arteries from orchidectomized rats. Furegrelate did not modify the EFS-induced NA release or vasoconstrictor response in arteries from either control or orchidectomized rats. The EFS-induced PGI2 release was not modified by furegrelate in arteries from control rats, but was increased in arteries from orchidectomized rats. The results of the present study show that endogenous male sex hormone deprivation i) increases non-endothelial TXA2 release and ii) regulates the effect of endogenous TXA2 on the EFS-induced response through different mechanisms that, at the least, involve the NO and PGI2 systems. In arteries from control rats, inhibition of TXA2 formation decreases the EFS-induced response by increasing neuronal NO release. In arteries from orchidectomized rats, the EFS-induced response is unaltered after the inhibition of TXA2 formation, by increasing PGI2 release.

Orchidectomy increases beta-adrenoceptor activation-mediated neuronal nitric oxide and noradrenaline release in rat mesenteric artery.

BACKGROUND/AIMS: A previous study has demonstrated that endogenous male sex hormones do not alter neuronal nitric oxide (NO) release in rat mesenteric artery. However, the regulatory role of endogenous male sex hormones on noradrenaline (NA) release in rat mesenteric artery is not known. The present study was designed to analyze whether endogenous male sex hormones influence the NA release induced by electrical field stimulation (EFS), as well as the possible modification in NA and neuronal NO release by presynaptic beta-adrenoceptor activation. METHODS: For this purpose, mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. Basal and EFS-induced neuronal NO and NA release, as well as the contractile effect induced by EFS, was measured. RESULTS: Basal and EFS-induced neuronal NO and NA release were similar in arteries from control and orchidectomized rats. The beta-adrenoceptor agonist clenbuterol did not modify EFS-induced neuronal NO and NA release in arteries from control rats. In contrast, in arteries from orchidectomized animals, clenbuterol increased both neuronal NO and NA release; this increase was prevented by incubation with the beta-adrenoceptor antagonist propranolol. However, the contractile response elicited by EFS was not modified by clenbuterol in either group of rats. CONCLUSIONS: These results show that orchidectomy does not alter the EFS-induced NA release. What is more, activation of presynaptic beta-adrenoceptors does not modify EFS-induced NA and neuronal NO release in arteries from control rats although it increases the release of both neurotransmitters in arteries from orchidectomized rats. Despite these modifications, the EFS-induced contractile response is preserved in arteries from orchidectomized rats.

Hidatidosis pulmonar en el hospital de Coquimbo / Lung hydatidosis at Coquimbo hospital

Se presenta la experiencia de 5 años en el tratamiento quirúrgico de la hidatidosis pulmonar, analizando la técnica quirúrgica y complicaciones. Se operaron 28 quistes hidatídicos en un total de 24 pacientes. Hubo 3 casos con quistes bilaterales. Se distribuían en 14 hombres y 10 mujeres, con una edad promedio de 39 años (rango de 16 a 76 años). Diecinueve pacientes eran sintomáticos, presentando tos y dolor torácico todos ellos. La técnica quirúrgica fue quistectomía y resección de la periquística en 19 casos, se le agregó capitonaje en 7 y en 2 se efectuó lobectomía. No hubo mortalidad en la serie

Desarrollo de la colangiografía endoscópica en un servicio de cirugía / Development of endoscopic cholangiography in a surgical service

La colangiopancreatografía endoscópica (CPRE), requiere considerable entrenamiento para realizarla efectivamente. Es el procedimiento de mayor riesgo que desarrollan los cirujanos endoscopistas, con morbilidad de 5 a 10 por ciento y mortalidad hasta 1 por ciento. En mayo de 1997, se diseña el protocolo de la experiencia. Se establece una marcha blanca (etapa 1), período que se termina al alcanzar un 70 por ciento de canulación. Entre mayo de 1997 y julio de 1999, se realizaron 201 CPRE; 52 en etapa 1 y 149 en etapa 2. El promedio de edad fue 62 años. Coledocolitiasis, ictericia obstructiva y colangitis fueron las principales indicaciones. La canulación en etapa inicial se logró en un 65 por ciento, alcanzando en la segunda etapa un 96 por ciento. La papilotomía estándar se logró en etapa inicial en un 27 , llegando a 80 por ciento en etapa 2. En la etapa 1, un 76 por ciento de los casos se terminó en el primer intento, en la etapa 2, esta cifra llegó al 92 por ciento. Las complicaciones observadas fueron hemorragia de papila 4 casos, retención de Dormia 1 y 2 pancreatitis aguda. La mortalidad registrada es de 0,5 por ciento. La colangiopancreatografía endoscópica (CPRE), requiere considerable entrenamiento para realizarla efectivamente. Es el procedimiento de mayor riesgo que desarrollan los cirujanos endoscopistas, con morbilidad de 5 a 10 por ciento y mortalidad hasta 1 por ciento. En mayo de 1997, se diseña el protocolo de la experiencia. Se establece una marcha blanca (etapa 1), período que se termina al alcanzar un 70 por ciento de canulación. Entre mayo de 1997 y julio de 1999, se realizaron 201 CPRE; 52 en etapa 1 y 149 en etapa 2. El promedio de edad fue 62 años. Coledocolitiasis, ictericia obstructiva y colangitis fueron las principales indicaciones. La canulación en etapa inicial se logró en un 65 por ciento, alcanzando en la segunda etapa un 96 por ciento. La papilotomía estándar se logró en etapa inicial en un 27 por ciento, llegando a 80 por ciento en etapa 2. En la etapa 1, un 76 por ciento de los casos se terminó en el primer intento, en la etapa 2, esta cifra llegó al 92 por ciento. Las complicaciones observadas fueron hemorragia de papila 4 casos, retención de Dormia 1 y 2 pancreatitis aguda. La mortalidad registrada es de 0,5 por ciento

Uso inmediato de la gastrostomía endoscópica / Immediate use of the endoscopic gastrostomy

Entre junio de 1996 y julio de 1997, se aplicó un protocolo prospectivo a todos los pacientes sometidos a gastrostomía endoscópica percutánea (GEP), indicándose el uso de la gastrostomía de inmediato, sin mediar espera ni prueba con soluciones isotóricas. El procedimiento fue realizado con la técnica de Ponsky, modificada por Kirberg, y el seguimiento se realizó hasta cumplir un mes. Se realizaron 41 GEP, 22 hombres y 19 mujeres. Al cumplir los 30 días de la instalación de la sonda, todos los pacientes la estaban usando sin problemas, 8 permanecían hospitalizados y 33 habían sido dados de alta. No se evidenció ninguna complicación atribuible al uso inmediato de la vía. En nuestra experiencia, el uso inmediato de la gastrostomía endoscópica percutánea es una indicación segura y médicamente prudente

A reinvestigation towards the conformation of methyl alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside by a combined ROE and molecular dynamics analysis.

Conformational analysis of alpha-D-Man p-(1-->6)-alpha-D-Man p1-OMe, by a combination of extensive molecular dynamics calculations in water and ROE buildup series, afforded two main minima, namely, phi/psi = 95/-178 and phi/psi = 140/-185. Transitions between these minima are observed, which have not previously been demonstrated using other approaches. In contrast to literature data for the glycosidic linkage, describing equal populations of both the gg and the gt rotamers, it was found that the gg conformer is present to ca. 96%. The non-reducing mannosyl unit showed approximately a 1:1 ratio for the gg:gt equilibrium, in accordance with earlier reports.

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.

Studies of the molecular recognition of synthetic methyl beta-lactoside analogues by Ricinus communis agglutinin.

The 2-, 3-, 6-, 2'-, 3'-, 4'-, and 6'-deoxy derivatives and the 3-O-methyl derivative of methyl beta-lactoside have been synthesised and their binding to the galactose-specific agglutinin from Ricinus communis (RCA-120) has been investigated. The results indicate that HO-3,4,6 of the beta-D-galactopyranose moiety are the key polar groups. The main difference from the closely related ricin lectin RCA-60 involves HO-6 of the D-glucopyranose moiety, which seems to contribute to the binding of the carbohydrate to RCA-60 but not to RCA-120.

4-O-beta-D-galactopyranosyl-D-xylose: a new synthesis and application to the evaluation of intestinal lactase.

4-O-beta-D-Galactopyranosyl-D-xylose (2) was prepared from benzyl 2,3-O-isopropylidene-beta-D-xylopyranoside by glycosylation with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide and subsequent deprotection. Compound 2 was hydrolyzed in vitro by intestinal lactase; the Vmax was 25% of that with lactose and the Km was 370mM (cf. 27mM for lactose). Oral administration of 2 suckling rats led to urinary excretion of D-xylose which could be estimated colorimetrically.

N.m.r. studies of the conformation of analogues of methyl beta-lactoside in methyl sulfoxide-d6.

The 1H- and 13C-n.m.r. spectra of solutions of methyl beta-lactoside (1), all of its monodeoxy derivatives (2, 3, 6-10), the 3-O-methyl derivative (4), and methyl 4-O-beta-D-galactopyranosyl-D-xylopyranoside (5) in methyl sulfoxide-d6 have been analysed. The n.O.e.'s and specific desheildings indicate similar distributions of low-energy conformers, comparable to those in aqueous solution. The major conformer has torsion angles phi H and psi H of 49 degrees and 5 degrees, respectively, with contributions of conformers with phi/psi 24 degrees/-59 degrees, 22 degrees/32 degrees, and 6 degrees/44 degrees.

The conformation of 1,6-anhydrolactose and its hexa-acetate in solution.

The conformation of 1,6-anhydrolactoase (1) has been investigated by n.m.r. spectroscopy and molecular mechanics calculations. For a solution in D2O, the 1,6-anhydroglucopyranoid ring has a 1C4 conformation, whereas there is a approximately 1:1 equilibrium between the 1C4 and the BO,3 conformations in (CD3)2SO. There is restricted flexibility with phi -80 +/- 20 degrees and psi -120 +/- 40 degrees. The hexa-acetate (2) of 1 shows a similar conformational behaviour.

Studies on the molecular recognition of synthetic methyl beta-lactoside analogs by ricin, a cytotoxic plant lectin.

The binding of methyl beta-lactoside and of all possible monodeoxy derivatives of methyl beta-lactoside to the galactose-specific highly cytotoxin lectin ricin, has been investigated. The distribution of low-energy conformers of the disaccharide structures has been first determined using molecular-mechanics calculations and high-resolution NMR spectroscopy. The nuclear Overhauser enhancements and specific deshieldings observed are in agreement with a similar distribution of low-energy conformers for all studied compounds which may be described by a major conformer defined by phi (H1'-C1'-O1'-C4) and psi (C1'-O1'-C4-H4) torsion angles of 49 degrees and 5 degrees, respectively, with contribution of conformers with angles phi/psi 24 degrees/-59 degrees, 22 degrees/-32 degrees and 6 degrees/-44 degrees. Assuming that the disaccharides bind to the lectin in these preferred conformations, the apparent dissociation constants for the ricin-disaccharide complexes have been interpreted in terms of specific polar and nonpolar interactions. In agreement with X-ray data, the hydroxyl groups at positions 3, 4 and 6 of the beta-D-galactopyranose moiety appear as key polar groups in the interaction with ricin. These results are in contrast to previous results which have established that position 6 is not involved in lectin binding. An important nonpolar interaction involving position 3 of the beta-D-glucopyranose moiety, seems to be operative. The distribution of low-energy conformers of these disaccharide structures permits this interaction to take place with the hydroxyl group at this position intramolecularly bonded, thus rendering this region of the molecule more lipophylic in character for acceptance into nonpolar regions of the combining site.

[Changes in nutritional and biochemical parameters in septic patients undergoing total parenteral nutrition. Their relationship with mortality]. / Evolución de parámetros nutricionales y bioquímicos en pacientes sépticos bajo NPT. Su relación con la mortalidad.

The response of 24 septic patients admitted into the ICU to total parenteral nutrition was studied, and the course of the parameters evaluated from non-surviving (14) and surviving patients was compared. Serum glucose, triglyceride, albumin, transferrin, prealbumin, RBP, zinc levels and CD4/CD8 ratio were measured at 48 hours, 4 days and 8 days after the onset of parenteral nutrition. Overall, a significant increase in prealbumin (p less than 0.01) and RBP (p less than 0.05) levels were the only findings to parenteral nutrition response. When the non-surviving and surviving groups were compared, the former showed a significant increase in serum triglyceride (p less than 0.001), while a significant increase in serum transferrin and CD4/CD8 ratio (p less than 0.01) was observed in the latter.