Reference miRNAs for colorectal cancer: analysis and verification of current data.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Relationship between pharmacokinetics and pharmacodynamics of clopidogrel in patients undergoing percutaneous coronary intervention: comparison between vasodilator-stimulated phosphoprotein phosphorylation assay and multiple electrode aggregometry.

UNLABELLED: ESSENTIALS: The reliability of platelet tests as markers of the variable bioavailability of clopidogrel is not yet defined. Kinetics of clopidogrel active metabolite (CAM) and platelet response were studied in ischemic heart disease. CAM plasma maximum concentration (Cmax ) predicted vasodilator-stimulated phosphoprotein (VASP-P). Timely performed VASP-P, not an aggregation-based test, may be a surrogate for clopidogrel bioavailability. BACKGROUND: The high inter-individual variability in the inhibition of platelet function by clopidogrel is mostly explained by high variability in its transformation to an active metabolite (CAM). Objective We investigated the relations between pharmacokinetics and pharmacodynamics of CAM by comparing two methods of platelet function. METHODS: We enrolled 14 patients undergoing percutaneous coronary interventions for non-ST-segment elevation acute coronary syndrome or inducible myocardial ischemia. Plasma concentrations of clopidogrel and CAM, phosphorylation of vasodilator-stimulated phosphoprotein (VASP-P), expressed as a platelet reactivity index (PRI) and whole-blood platelet aggregation (multiple electrode aggregometer, MEA) were measured before and after a 600-mg clopidogrel loading dose (nine time-points) and before and after 75-mg maintenance doses on days 2, 7 and 30. RESULTS: Plasma concentrations of clopidogrel and CAM were highly variable. CAM reached maximal concentration (Cmax ) (median, 110.8 nm; range, 41.9-484.8) 0.5-2 h after the loading dose. A sigmoid dose-response curve defined the relations between CAMCmax and PRI after 3 to 24 h (IC50 , 459.6 nm; 95% confidence interval, 453.4-465.7; R(2) = 0.82). PRI was unchanged from baseline in patients with the lowest CAMCmax (< 83 nm, n = 7), indicating low sensitivity of VASP-P. PRI values were also predicted by CAMCmax at days 2, 7 and 30. Platelet aggregation measured by MEA did not show significant relations with either PRI or with CAM pharmacokinetics at any time-point. CONCLUSIONS: After 600 mg clopidogrel, VASP-P, but not whole-blood platelet aggregation measured by MEA, is almost entirely predicted by CAMCmax . VASP-P could be useful in studies aimed at investigating relations between CAM bioavailability and clinical events.

Epigenetic alteration: new insights moving from tissue to plasma - the example of PCDH10 promoter methylation in colorectal cancer.

BACKGROUND: Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features. METHODS: A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay. RESULTS: The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3-97.8%) and 5.9% (0-80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234). CONCLUSION: Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

Optimisation of radiological protocols for chest imaging using computed radiography and flat-panel X-ray detectors.

PURPOSE: Digital radiography technology has replaced conventional screen-film systems in many hospitals. Despite the different characteristics of new detector materials, frequently, the same radiological protocols previously optimised for screen film are still used with digital equipment without any critical review. This study addressed optimisation of exposure settings for chest examinations with digital systems, considering both image quality and patient dose. MATERIALS AND METHODS: Images acquired with direct digital radiography equipment and a computed radiography system were analysed with specially developed commercial software with a four-alternative forced-choice method: the most promising protocols were then scored by two senior radiologists. RESULTS: Digital technology offers a wide dynamic range and the ability to postprocess images, allowing use of lower tube potentials in chest examinations. The computed radiography system showed both better image quality and lower dose at lower energies (85 kVp and 95 kVp) than those currently used (125 kVp). Direct digital radiography equipment confirmed both its superior image quality and lower dose requirements compared with the storage phosphor plate system. CONCLUSIONS: Generally, lowering tube potentials in chest examinations seems to allow better image quality/effective dose ratio when using digital equipment.

Cirugía colorrectal laparoscópica: Resultados a corto plazo en una serie de 300 pacientes / Laparoscópica recto colonic surgery: Short term results in a series of 300 patients

Antecedentes: La cirugía laparoscópica ha modificado el manejo de la patología colorrectal. Objetivo: Analizar los resultados a corto plazo obtenidos con el abordaje laparoscópico de la patología colorrectal. Lugar de aplicación: Sección de coloproctología, Hospital Privado. Diseño: Cohorte, retrospectivo. Población: 300 pacientes (mediana de edad 62 años, 51,4% de mujeres) tratados mediante abordaje laparoscópico entre marzo de 2003 y abril 2007. Método: Análisis de una base de datos prospectiva. Conversión, readmisión hospitalaria y complicaciones posoperatorias fueron analizadas en base a las características demográficas, tipo de procedimiento efectuado y curva de aprendizaje. Se realizó un análisis multivariado para detectar factores asociados a conversión. Resultados: Conversión global: 15%. La resección anterior mostró un índice de conversión significativamente mayor al del resto de los procedimientos (29%, p=2.02). No observamos diferencias a lo largo de nuestra curva de aprendizaje, a excepción de una disminución del tiempo operatorio. Estadía hospitalaria: 3 días. Morbilida global: 19,6%. Las complicaciones postoperatorias más frecuentes fueron el íleo (9%), la infección de herida (2,3%) y la fístula anastomótica (1,3%). Mortalidad: 0.6%. Los hombres se convirtieron del doble que las mujeres (p=0.01) y se complicaron más (p=0.006). El sexo, el IMC y el tipo de procedimiento resultaron ser actores asociados a conversión. Conclusiones: La cirugía laparoscópica ha logrado trasladar los beneficios del abordaje nininvasivo al manejo de la patología colorrectal. Distintos factores pueden modificar sus resultados a corto plazo, especialmente en términos de conversión. El conocimiento de éstos puede contribuir a una adecuada selección de los casos.(AU)

Cirugía colorrectal laparoscópica: Resultados a corto plazo en una serie de 300 pacientes / Laparoscópica recto colonic surgery: Short term results in a series of 300 patients

Antecedentes: La cirugía laparoscópica ha modificado el manejo de la patología colorrectal. Objetivo: Analizar los resultados a corto plazo obtenidos con el abordaje laparoscópico de la patología colorrectal. Lugar de aplicación: Sección de coloproctología, Hospital Privado. Diseño: Cohorte, retrospectivo. Población: 300 pacientes (mediana de edad 62 años, 51,4% de mujeres) tratados mediante abordaje laparoscópico entre marzo de 2003 y abril 2007. Método: Análisis de una base de datos prospectiva. Conversión, readmisión hospitalaria y complicaciones posoperatorias fueron analizadas en base a las características demográficas, tipo de procedimiento efectuado y curva de aprendizaje. Se realizó un análisis multivariado para detectar factores asociados a conversión. Resultados: Conversión global: 15%. La resección anterior mostró un índice de conversión significativamente mayor al del resto de los procedimientos (29%, p=2.02). No observamos diferencias a lo largo de nuestra curva de aprendizaje, a excepción de una disminución del tiempo operatorio. Estadía hospitalaria: 3 días. Morbilida global: 19,6%. Las complicaciones postoperatorias más frecuentes fueron el íleo (9%), la infección de herida (2,3%) y la fístula anastomótica (1,3%). Mortalidad: 0.6%. Los hombres se convirtieron del doble que las mujeres (p=0.01) y se complicaron más (p=0.006). El sexo, el IMC y el tipo de procedimiento resultaron ser actores asociados a conversión. Conclusiones: La cirugía laparoscópica ha logrado trasladar los beneficios del abordaje nininvasivo al manejo de la patología colorrectal. Distintos factores pueden modificar sus resultados a corto plazo, especialmente en términos de conversión. El conocimiento de éstos puede contribuir a una adecuada selección de los casos.

Mutación del gen BMPR1A (Bone Morphogenetic Protein Receptor 1A) en un caso de poliposis juvenil / BMPR1A (Bone Morphogenetic Protein Receptor 1A) gene mutation in juvenile polyposis: a case report

Antecedentes: la poliposis juvenil (PJ) es una infrecuente afección hereditaria autosómica dominante caracterizada por la presencia de múltiples pólipos hamartomatosos gastrointestinales. Hasta el momento se han identificado 3 genes relacionados a esta afección: SMAD4 (cromosoma 18q21), el PTEN (cromosoma 10q23) y recientemente el BMPR1A (cromosoma 10q22-23). El diagnóstico genético permite optimizar el manejo de estos pacientes. Objetivo: presentar los resultados del diagnóstico clínico de poliposis juvenil. Método: paciente de sexo masculino de 16 años de edad con pólipos colónicos cuyas biopsias preoperatorias informaron la presencia de componentes adenomatosos, hamartosos e hiperplásicos. Luego de la resección endoscópica de 6 pólipos rectosigmoideos, se le realizó una colectomía subtotal con ileo-recto anastomosis. Antes de poder contar con el diagnóstico genético y a fin de determinar la posible afectación fenotípica se indicó videocolonoscopías (VFCC) a ambos padres y a cuatro hermanos. Luego del asesoramiento genético se obtuvo el consentimiento informado y se mandaron las muestras de sangre del paciente y sus padres a la Universidad de Iowa, USA para la determinación de mutaciones germinales en los genes SMAD 4 y BMPR1A. Resultados: todas las VFCC efectuadas fueron normales. El estudio molecular encontró una mutación germinal del gen BMPR1A (864-868 del ACTTGIVS7 + 1-2delgt) en el paciente y ausencia de la misma en ambos padres. Se concluyo que se trataba de una mutación "de novo" asociada a la poliposis juvenil y que por lo tanto ninguno de sus familiares presentaba riesgo aumentado. En base a esta información no se recomendó continuar con la vigilancia estricta de los mismos. Conclusión: la identificación de la mutación germinal permitió confirmar el diagnóstico de poliposis juvenil y estimar el riesgo de presentar dicha enfermedad en los familiares cosanguineos optimizando la estrategia de prevención en la familia. (AU)

Mutación del gen BMPR1A (Bone Morphogenetic Protein Receptor 1A) en un caso de poliposis juvenil / BMPR1A (Bone Morphogenetic Protein Receptor 1A) gene mutation in juvenile polyposis: a case report

Antecedentes: la poliposis juvenil (PJ) es una infrecuente afección hereditaria autosómica dominante caracterizada por la presencia de múltiples pólipos hamartomatosos gastrointestinales. Hasta el momento se han identificado 3 genes relacionados a esta afección: SMAD4 (cromosoma 18q21), el PTEN (cromosoma 10q23) y recientemente el BMPR1A (cromosoma 10q22-23). El diagnóstico genético permite optimizar el manejo de estos pacientes. Objetivo: presentar los resultados del diagnóstico clínico de poliposis juvenil. Método: paciente de sexo masculino de 16 años de edad con pólipos colónicos cuyas biopsias preoperatorias informaron la presencia de componentes adenomatosos, hamartosos e hiperplásicos. Luego de la resección endoscópica de 6 pólipos rectosigmoideos, se le realizó una colectomía subtotal con ileo-recto anastomosis. Antes de poder contar con el diagnóstico genético y a fin de determinar la posible afectación fenotípica se indicó videocolonoscopías (VFCC) a ambos padres y a cuatro hermanos. Luego del asesoramiento genético se obtuvo el consentimiento informado y se mandaron las muestras de sangre del paciente y sus padres a la Universidad de Iowa, USA para la determinación de mutaciones germinales en los genes SMAD 4 y BMPR1A. Resultados: todas las VFCC efectuadas fueron normales. El estudio molecular encontró una mutación germinal del gen BMPR1A (864-868 del ACTTGIVS7 + 1-2delgt) en el paciente y ausencia de la misma en ambos padres. Se concluyo que se trataba de una mutación "de novo" asociada a la poliposis juvenil y que por lo tanto ninguno de sus familiares presentaba riesgo aumentado. En base a esta información no se recomendó continuar con la vigilancia estricta de los mismos. Conclusión: la identificación de la mutación germinal permitió confirmar el diagnóstico de poliposis juvenil y estimar el riesgo de presentar dicha enfermedad en los familiares cosanguineos optimizando la estrategia de prevención en la familia.

Rectal cancer: local recurrence after surgery without radiotherapy.

PURPOSE: This study was designed to assess the local recurrence rate and prognostic factors for local recurrence in patients undergoing curative anterior or abdominoperineal resections without radiotherapy. METHODS: From January 1980 to December 1996, 514 consecutive patients underwent curative resections for rectal cancer. We excluded those with preoperative radiotherapy (n = 23), postoperative radiotherapy (n = 27), local resection (n = 36), and 11 (2.1 percent) patients who died postoperatively. The remaining 417 patients (249 males) with a median age of 64 (range, 21-90) years were analyzed. For upper third lesions, mesorectal tissue was excised down to at least 5 cm below the tumor. Total mesorectal excision was performed for lower and middle tumors. Postoperative chemotherapy was limited to patients with Stage III lesions. Median follow-up (and 95 percent confidence interval) was (5.2 4.3-5.9) years, with 87.7 percent of patients followed up longer than 24 months. Local recurrence was defined as any recurrence within the field of resection, regardless of the presence or absence of distant metastasis. RESULTS: Five-year local recurrence rate(and 95 percent confidence interval) was 9.7 (6.4-13) percent, with a median time to diagnosis of 15 (10-23) months. Local recurrence rates in Stages I, II, and III were: 3.1, 4.1, and 24.1 percent, respectively (P < 0.0001). In relation to node status, local recurrence rates were N0, 4.1 (1.7-6.5) percent; N1, 12.6 (4.6-20.6) percent; N2, 32.1 (12.1-52.1) percent; and N3, 59.3 (22.5-96.1) percent; (P < 0.00001). Lower third tumors had a higher local recurrence rate than middle and upper third tumors: 17.9, 7.1, and 5.1 percent, respectively (P = 0.002). Adjusted by stage, this difference was maintained only in Stage III tumors. Among lower tumors, those at 6 and 7 cm from the anal verge had a lower local recurrence rate than those below 6 cm (6.7 vs. 26.2 percent, respectively; P = 0.02). Accidental rectal perforation at or near the tumor site occurred in 12 cases (2.9 percent), showing a strong correlation with local recurrence (P < 0.0001). Multivariate analysis showed significant higher risk for lower third tumors (hazard ratio, 2.98) and positive nodes (hazard ratio, 4.78). CONCLUSIONS: Appropriate surgery without irradiation achieves excellent local control in N0 rectal cancers. Node metastasis, lower third localization (especially below 6 cm), and accidental rectal perforation at or near the tumor site are significantly associated with a higher local recurrence rate.

Management of a high-output postoperative enterocutaneous fistula with a vacuum sealing method and continuous enteral nutrition.

A postoperative enterocutaneous fistula is one of the most complex medical problems. Its treatment may become long-lasting, wearisome, and its outcome often is disappointing. Here, we describe the use of a novel device to treat a 67-year-old patient with a postoperative, high-output enterocutaneous fistula. A semipermeable barrier was created over the fistula by vacuum packing a synthetic, hydrophobic polymer covered with a self-adherent surgical sheet. To set up the system, we constructed a vacuum chamber equipped with precision instruments that supplied subatmospheric pressures between 350 and 450 mm Hg. The intestinal content was, thus, kept inside the lumen, restoring bowel transit and physiology. The fistula output was immediately reduced from a median of 800 ml/day (range, 400-1,600 ml/day), to a median of 10 ml/day (range, 0-250 ml/day), which was readily collected by the apparatus. Oral feeding was reinitiated while both parenteral nutrition and octreotide were withdrawn. No septic complications occurred, and the perifistular skin stayed protected from irritating intestinal effluents. Both the fistula orifice and the wound defect fully healed after 50 days of treatment. We believe this method may serve as a useful tool to treat selected cases of high-output enterocutaneous fistulas without the need for octreotide or parenteral nutrition.

[In vivo dosimetry and radiographic confirmation in radiotherapy of Hodgkin's lymphoma]. / Dosimetria in vivo e verifica radiografica nella radioterapia del linfoma di Hodgkin.

In September 1991 a protocol for quality control of large shaped irradiation fields was started in our department. In vivo dosimetry with semiconductor detectors was used to measure the absorbed dose and patient positioning was checked with portal films weekly. First, we set a computed dosimetric system yielding dosimetric values in real time and allowing their easy storage. Then, we calibrated the diodes and determined the correction factors for each of them outside standard conditions. Entrance dose, exit dose and midline dose were measured in 62 patients undergoing supradiaphragmatic radiation therapy for Hodgkin's lymphoma. The exist dose was measured weekly to assess treatment repeatability. High agreement was observed between measured and calculated doses; repeatability was also high, since only 6% of exit dose measurements exceeded 5% of the first determination. In 33 patients portal films were obtained in the first treatment session, and thereafter weekly, to assess mispositioning relative to simulation (reproducibility) and from one session to another (repeatability). A small systematic error was detected in both longitudinal (x = -3 mm; SD = 3.7 mm) and transverse (x = -2 mm; SD = 3.4 mm) directions. Statistically significant errors (> 6 mm) were observed in 14% of patients. Reproducibility was excellent. The protocol reported on in this paper not only helps avoid systematic dosimetric and/or positioning errors in the patients, but also helps identify the main causes of uncertainty and thus remove them.

Single-dose oral ciprofloxacin plus parenteral metronidazole for perioperative antibiotic prophylaxis in colorectal surgery.

From 1983 until 1990 our standard antibiotic prophylaxis in colorectal surgery has been 80 mg gentamicin plus 500 mg metronidazole, both administered intravenously at the beginning of anesthesia, and then 80 mg gentamicin every 8 h for 3 days. In this time period, 718 patients undergoing elective colorectal surgery by abdominal approach were treated with this prophylactic procedure. The mean wound infection rate was 13.4% with no significant differences between years. In the present study, including 100 adult patients, intravenous gentamicin has been replaced by a single oral dose of 750 mg ciprofloxacin randomly administered 1, 2 or 3 h (+/- 30 min) before anesthesia. All other procedures remained unchanged including intravenous metronidazole. In order to correlate an eventual infection with the actual tissue concentrations during surgery, samples of colon, peritoneum, muscle, subcutaneous fat and skin were obtained from all patients and assayed by HPLC. In only 3 cases (3% of them casuistic) was a wound infection observed, 2 of minor importance and 1 case with wound dehiscence. This infection rate is significantly lower than 13.4% obtained with our previous standard regimen, and lower than the 9% wound infection rate considered to be the 'gold standard' for elective colon resections. The effective ciprofloxacin concentrations in tissues were usually notably higher than the MICs of the pathogens commonly observed in surgical infections of colorectal surgery. By far the highest ciprofloxacin concentrations were observed in colon tissue which may explain the absence of anastomotic dehiscence or peritoneal sepsis in our series.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthropomorphic robotics. I. Representing mechanical complexity.

A study of the fundamental principles upon which manipulation dexterity is based cannot help mixing robotic and neurophysiological concepts. A preliminary step in this study consists of trying to understand the complexity of manipulation dynamics. Though complexity shows itself in the massive number of elements of kinematic and dynamic equations, the fundamental simplicity of the underlying mechanical laws suggests to look for a structure, particularly from the computational point of view. Accordingly, a working computational model is proposed that organizes the massive computational load into a structure which is composed of a small number of computational units and lends itself to parallel computation.

Anthropomorphic robotics. II. Analysis of manipulator dynamics and the output motor impedance.

An important factor in trying to capture the complexity of many manipulation problems is the notion of Output Motor Impedance, i.e., the relationship between a set of disturbing forces and the resulting variation in arm configuration. The functional significance of such force/displacement characteristics is investigated, showing how several aspects of different manipulation tasks (holding against gravity, inserting, fast moving, and throwing) can be naturally described in terms of appropriate modulation of the impedance characteristics of the manipulator. For this reason, impedance modulation can be considered an integral part of motor control.