Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer.

BACKGROUND: A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse. PATIENTS AND METHODS: One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR. RESULTS: NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r = 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P = 0.0014 and HR, 11.33; log-rank P = 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5 months. CONCLUSIONS: Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.

Anastomotic leakage after colon cancer resection: does the individual surgeon matter?

AIM: Anastomotic leakage is one of the most feared complications after colonic resection. Many risk factors for anastomotic leakage have been reported, but the impact of an individual surgeon as a risk factor has scarcely been reported. The aim of this study was to assess if the individual surgeon is an independent risk factor for anastomotic leakage in colonic cancer surgery. METHOD: This was a retrospective analysis of prospectively collected data from patients who underwent elective resection for colon cancer with anastomosis at a specialized colorectal unit from January 1993 to December 2010. Anastomotic leaks were diagnosed according to standardized criteria. Patient and tumour characteristics, surgical procedure and operating surgeons were analysed. A logistic regression model was used to discriminate statistical variation and identify risk factors for anastomotic leakage. RESULTS: A total of 1045 patients underwent elective colon cancer resection with primary anastomosis. Anastomotic leakage occurred in 6.4% of patients. Ileocolic anastomosis had an anastomotic leakage rate of 7.2%, colo-colonic/colorectal anastomosis 5.2% and ileorectal anastomosis 12.7%, with intersurgeon variability. The independent risk factors associated with anastomotic leakage were the use of perioperative blood transfusion (OR 2.83, CI 1.59-5.06, P < 0.0001) and the individual surgeon performing the procedure (OR up to 8.44, P < 0.0001). CONCLUSION: In addition to perioperative blood transfusion, the individual surgeon was identified as an important risk factor for anastomotic leakage. Efforts should be made to reduce performance variability amongst surgeons.

Anaplasmataceae in wild ungulates and carnivores in northern Spain.

Wild vertebrates are essential hosts for tick-borne diseases but data on the prevalence and diversity of Anaplasma spp. in wildlife are scarce. In this study, we used real-time PCR to investigate the distribution of Anaplasma species in spleen samples collected from 625 wild animals (137 cervids, 227 wild boar, and 261 carnivores) in two regions in northern Spain. A first generic real-time PCR assay was used to screen for the presence of Anaplasma spp. followed by a second species-specific multiplex real-time PCR or partial sequencing of the 16S rRNA gene for species identification. Anaplasma phagocytophilum was highly prevalent in cervids (64.2%), but it was absent from wild boar and carnivores. Interestingly, Anaplasma marginale and Anaplasma ovis were not detected in cervids, but Anaplasma centrale was identified in 1 roe deer and 1 red deer, A. bovis in 4 roe deer, and a novel Ehrlichia sp. in one badger. These findings were highly associated with the tick burden identified in the different hosts. Thus, Ixodes ricinus, the recognized vector of A. phagocytophilum in Europe, was the main tick species parasitizing cervids (93.5%, 1674/1791), whereas Dermacentor reticulatus was the most abundant in wild boar (76.1%, 35/46) and Ixodes hexagonus in carnivores (58.4%, 265/454). More investigations are needed to assess the impact of the different Anaplasma species in wildlife and the risk of transmission to domestic animals.

Concomitance and interactions of pathogens in the Iberian wolf (Canis lupus).

With the aim of improving our understanding of their epidemiological features, exposure to or presence of Canine Parvovirus (CPV), Canine Distemper Virus (CDV), Leishmania infantum and Sarcoptes scabiei were studied in 88 wild wolves from Asturias (Northern Spain) by means of long-term (2004-2010) serological and molecular data. Individual and population factors and the possible interactions between them were also statistically analyzed for better understanding the contact/presence of studied pathogens. The overall seroprevalence values were 19%, 61%, 20% and 0% for CDV, CPV, S. scabiei and Leishmania, respectively, while a 46% of studied wolves showed Leishmania genetic material presence. Sarcoptic mange, CDV and CPV showed higher seroprevalence values in the areas with higher wolf densities, and a positive association between CDV and S. scabiei antibody responses was detected. Reported data highlight the need of considering concomitant pathogens and their possible interactions for a better understanding of diseases and their management in wildlife.

[Hydroxyethyl starch to protect renal function in laparoscopic surgery]. / Uso de hidroxietil almidón (Voluven) como protección de la función renal en cirugía laparoscópica.

OBJECTIVE: To evaluate the effect of prehydration with hydroxyethyl starch 130/0.4 (Voluven) compared to lactated Ringer solution in laparoscopic cholecystectomy. PATIENTS AND METHODS: We performed a randomized single-blind clinical trial on patients classified as ASA 1 and 2. The exclusion criteria were hypertension, kidney failure, treatment with diuretics or other antihypertensive drugs, diabetes, and use of nonsteroidal anti-inflammatory drugs. Balanced general anesthesia with remifentanil and sevoflurane was used. The total volume of administered fluids (including prehydration) was 2 mL x kg(-1) x h(-1) fasting plus 5 mL x kg(-1) x h(-1) during surgery. Group 1 was prehydrated 30 minutes before surgery with 500 mL of lactated Ringer solution in group 1; in group 2 the same quantity of Voluven was used. Ringer solution was used in both groups to provide additional fluids. Blood pressure was kept within 20% above or below baseline values. Standard anesthetic monitoring was performed. Intraoperative diuresis and creatinine clearance were recorded. The groups were compared using the t test; a P value of 05 or less was considered significant. RESULTS: Twenty-nine patients were enrolled in the study. Diuresis and creatinine clearance were significantly higher in the group that received prehydration with Voluven. The mean (SD) creatinine clearance rate was 176.44 (1433) mL x min(-1) in group 1 and 61.90 (6.6) mL x min(-1) in group 2 (P = .036). The mean volume of urine excreted was 1.71 (0.06) mL x kg(-1) x h(-1) in group 1 and 0.47 (0.02) mL x kg(-1) x h(-1) in group 2 (P = .017). CONCLUSION: Prehydration with Voluven can be an effective measure for protecting renal function against the adverse effects of pneumoperitoneum in laparoscopic surgery.

Uso de hidroxietil almidón (Voluven®) como protecciónde la función renal en cirugía laparoscópica / Hydroxyethyl starch to protect renal function in laparoscopic surgery

OBJETIVO: Valorar el efecto sobre la función renal dela prehidratación con hidroxietil almidón 130/0,4 (Voluven®) frente al Ringer Lactato en las colecistectomíaslaparoscópicas.PACIENTES Y MÉTODOS: Ensayo clínico aleatorizadosimple ciego en pacientes ASA I, II. Criterios de exclusión:hipertensión arterial, insuficiencia renal, tratamientocon diuréticos u otros antihipertensivos, diabéticos,uso de anti-inflamatorios no esteroideos. Anestesiageneral balanceada basada en remifentanilo-sevofluorano.Volumen total de fluidos administrados: (incluida laprehidratación): 2 mL Kg–1 h–1 ayuno + 5 mL Kg–1 h–1 deintervención. Prehidratación 30 minutos antes de laintervención con 500 mL de Ringer Lactado (RL) en elGrupo I y con la misma cantidad de Voluven® en el GrupoII. Resto de la fluidoterapia basada en Ringer Lactatoen ambos grupos. Mantenimiento de la presión arterialmedia ± 20% de los valores iniciales. Monitorizaciónanestésica habitual. Medida de diuresis intraoperatoriay aclaramiento de creatinina de diuresis intraoperatoria(ClCr). Comparación de grupos mediante t Student conun nivel de significación p ≤ 0,05.RESULTADOS: Se incluyeron 29 pacientes. Las diuresisy el aclaramiento de creatinina obtenidos fueron mayoresde forma estadísticamente significativa en el grupoen el que se realizó la prehidratación con Voluven®. ClCr(mL min–1) GRUPO I: 176,44 ± 14,33, GRUPO II:61,90 ± 6,6 (p = 0,036); Diuresis (mL Kg–1 h–1) GRUPO I:1,71 ± 0,06, GRUPO II 0,47 ± 0,02 (p = 0,017).CONCLUSIONES: La prehidratación con Voluven® puedeser una medida eficaz en la protección de la función renalfrente a los efectos adversos del neumoperitoneo en lacirugía laparoscópica (AU)

OBJETIVE: To evaluate the effect of prehydration withhydroxyethyl starch 130/0.4 (Voluven) compared tolactated Ringer solution in laparoscopic cholecystectomy.PATIENTS AND METHODS: We performed a randomizedsingle-blind clinical trial on patients classified as ASA 1and 2. The exclusion criteria were hypertension, kidneyfailure, treatment with diuretics or other antihypertensivedrugs, diabetes, and use of nonsteroidal anti-inflammatorydrugs. Balanced general anesthesia with remifentanil andsevoflurane was used. The total volume of administeredfluids (including prehydration) was 2 mL·kg-1·h-1 fastingplus 5 mL·kg-1·h-1 during surgery. Group 1 wasprehydrated 30 minutes before surgery with 500 mL oflactated Ringer solution in group 1; in group 2 the samequantity of Voluven was used. Ringer solution was used inboth groups to provide additional fluids. Blood pressurewas kept within 20% above or below baseline values.Standard anesthetic monitoring was performed.Intraoperative diuresis and creatinine clearance wererecorded. The groups were compared using the t test; a Pvalue of 05 or less was considered significant.RESULTS: Twenty-nine patients were enrolled in thestudy. Diuresis and creatinine clearance weresignificantly higher in the group that receivedprehydration with Voluven. The mean (SD) creatinineclearance rate was 176.44 (14.33) mL·min-1 in group 1and 61.90 (6.6) mL·min-1 in group 2 (P=.036). The meanvolume of urine excreted was 1.71 (0.06) mL·kg-1·h-1 ingroup 1 and 0.47 (0.02) mL·kg-1·h-1 in group 2 (P= .017).CONCLUSION: Prehydration with Voluven can be aneffective measure for protecting renal function againstthe adverse effects of pneumoperitoneum in laparoscopicsurgery (AU)

The ideal lateral internal sphincterotomy: clinical and endosonographic evaluation following open and closed internal anal sphincterotomy.

OBJECTIVE: To evaluate the relationship between extent of internal sphincter division following open and closed sphincterotomy, as assessed by anal endosonography, with fissure persistence/recurrence and faecal incontinence. METHOD: A total of 140 consecutive patients undergoing lateral internal sphincterotomy (LIS) for idiopathic chronic anal fissure were prospectively studied. Preoperative clinical assessment was performed together with a postoperative clinical and endosonographic examination. Three zones of the internal sphincter, identifiable by endosonography, were used to describe the uppermost extent of LIS. Primary end-points were fissure persistence/recurrence and faecal incontinence. RESULTS: A total of 140 patients, median age 49.5 years (IQR: 38-56 years) were included. Seventy-five (53.6%) and 65(46.4%) patients underwent percutaneous LIS (PLIS) and open LIS (OLIS) respectively. Median follow-up was 21 months (IQR: 14-29 months). Persistence and recurrence rates were 2.9% (4/140) and 5.7% (8/140) respectively. 7.9% (11/140) patients scored > 3 on the Jorge and Wexner Faecal Incontinence scale. PLIS was associated with a trend towards higher fissure persistence/recurrence rates than OLIS (12.0%vs 4.6%, P = 0.141). OLIS was significantly associated with a higher proportion of complete sphincterotomies (CS) than PLIS (56/65 vs 48/75, P = 0.003). A CS was associated with a lower fissure persistence or recurrence rate (1/104 vs 11/36, P < 0.001) but higher incontinence scores (11/104 vs 0/36 cases with Wexner scores > 3, P = 0.042) than following incomplete sphincterotomy. There was a strongly significant increase in incontinence scores (P < 0.001) and decrease in recurrence rates (P < 0.001) with increasing length of sphincterotomy. CONCLUSION: We recommend a short and CS using either PLIS or OLIS for the treatment of idiopathic anal fissure.

Management of intersphincteric abscesses.

OBJECTIVE: Intersphincteric abscesses are relatively rare, and in some cases of upward extensions in the supralevator plane, can be difficult to manage. The aim of this study was to analyse the type of treatment used in these abscesses. METHODS: Twenty-one intersphincteric abscesses treated by endoanal drainage in our colorectal unit between 1992 and 2004 were reviewed from our database; location and extension of the abscess, type of treatment and recurrence rates and the use of endoanal ultrasound were studied. RESULTS: Ninety per cent of patients were male; 10 had a previous history of surgery for perianal abscess and suppuration (48%); 16 (76%) had a posterior location and five were anterolateral. Twelve patients had low intersphincteric abscesses and were treated by laying open the abscess and dividing the internal sphincter. Nine were found to have high extensions into the intermuscular planes and were treated by staged procedures: a temporary transanal mushroom catheter was used in seven patients. Endoanal ultrasound was used initially in seven patients (33.3%) and for the evaluation of definitive treatment in 11 (52%). CONCLUSIONS: Low intersphincteric abscesses should be treated by de-roofing of the abscess and division of the internal sphincter up to a level of the dentate line. High intersphincteric abscesses are relatively frequent and mostly require staged surgery with a temporary mushroom (de Pezzer) catheter. Accurate anatomical ultrasound localization and proper drainage become important to avoid recurrences or extrasphincteric fistulas.

Pathological features in marine birds affected by the prestige's oil spill in the north of Spain.

A total of 2,465 seabirds, mainly common murres (Uria aalge), razorbills (Alca torda), and puffins (Fratercula arctica) that beached in the northwestern part of Spain after the "Prestige" oil spill on 19 November 2002 were examined by pathological methods. Birds were divided into three groups: dead birds with the body covered (group 1) or uncovered (group 2) by oil and birds recovered alive but which died after being treated at a rescue center (group 3). The main gross lesions were severe dehydration and emaciation. Microscopically, hemosiderin deposits, related to cachexia and/or hemolytic anemia, were observed in those birds harboring oil in the intestine. Severe aspergillosis and ulcers in the ventriculus were found only in group 3 birds, probably because of stress associated with attempted rehabilitation at the rescue center. The mild character of the pathological changes suggests that petroleum oil toxicosis causes multiple sublethal changes that have an effect on the ability of the birds to survive at sea, especially weak and young, inexperienced animals. Dehydration and exhaustion seem to be the most likely cause of death.

A prospective audit of the complications of loop ileostomy construction and takedown.

AIM: A prospective review of the complications of ileostomy construction and takedown. MATERIALS AND METHODS: One hundred twenty-seven consecutive patients undergoing construction of a loop ileostomy were included in a prospective nonrandomized computer database. Complications of the loop ileostomy were assessed prior to and after closure. Three closure techniques were performed [enterotomy suture (25.7%), resection and hand sewn (31.2%) or stapled anastomosis (43.1%)] and compared. RESULTS: One hundred twenty-seven (73 male, 54 female) patients, mean age 54 years were included from 1992 to 2002. Seventy-two patients underwent anterior resection for low rectal carcinoma, 30 an ileoanal pouch for ulcerative colitis and 25 for miscellaneous conditions. Fifty-nine pre-takedown complications occurred in 50 (39.4%) patients. The most common were dermatitis (12.6%) and erythema (7.1%). The most severe were dehydration in 1 patient and stomal prolapse in 4 patients. Closure was associated with a complication rate of 33.1% and a mortality rate of 0.9%. Wound infection occurred in 18.3% and small bowel obstruction in 4.6%. Anastomotic leak requiring reanastomosis occurred in 2.8% and enterocutaneous fistula treated conservatively in 5.5%. There were no statistically significant differences in morbidity between closure techniques (p = 0.892). There were no statistically significant differences in complications (p = 0.516) between patients with ulcerative colitis and those with neoplasia (39.29% vs. 32.2%). CONCLUSIONS: Loop ileostomy construction and takedown is associated with considerable morbidity, mostly minor. No differences exist between technique used for closure or the baseline pathology of the patient.

Comparison between two warm ischemic models in experimental liver transplantation in pigs.

BACKGROUND: Experimental models of warm ischemia in liver transplantation have been employed to study the mechanisms and treatment of ischemia reperfusion injury. METHODS: We compared a control group without (group A, n = 10) versus two models of warm ischemia of liver transplants in pigs: namely, occlusion of the hepatic artery and portal vein for 30 minutes (group B, n = 23) and extraction of the liver 60 minutes after cardiac arrest (group C, n = 5). Liver function tests, coagulation studies, and liver biopsies were performed during the first 24 hours post-liver transplant. RESULTS: Clamping of the hepatic vasculature in group B produced a significant liver injury compared with the control group: elevation of the ALT and an abnormal 1-hour post-revascularization biopsy similar to that observed in the cardiac arrest group C. The transaminase levels were lower among group A animals (P <.05). But the hepatic synthetic functions as reflected in the protrombin time (PT) were not affected in group B versus group A. The alteration in PT with respect to the initial value was similar among group A and group B animals, which were significantly less than that in group C (P <.05). CONCLUSIONS: Occlusion of the hepatic artery and portal vein, a simple surgical maneuver, causes moderate damage to a liver graft but less alteration of hepatic synthetic function. Clamping of the hepatic vasculture obtains more long-term survivors after OLT than cardiac arrest.

Impact of surgeon organization and specialization in rectal cancer outcome.

PURPOSE: The present study was designed to assess the differences in the outcome of patients with rectal cancer treated by a group of surgeons before and after being organized as a Coloproctology Unit at the same University Department of Surgery. METHODS: Comparison of two periods of rectal cancer surgery: I (1986-91) and II (1992-95). Period I: 94 patients were operated on by 14 general surgeons. Period II: 108 patients were operated on by only 4 surgeons of the same group organized as a Colorectal Surgery Unit after visiting referral centres abroad, adopting techniques such as total mesorectal excision (TME) for middle and low rectal cancer and washout of rectal stump. Mean follow-up during periods I and II was 69.1 and 42.0 months, respectively. A prospective data base analysis was used. Survival and local recurrence rates were calculated by the actuarial method. For comparison between groups the log rank method was used. RESULTS: The two groups were comparable with respect to mean age, gender, TNM and rectal tumour location. A significant increase in radical resectability and a decrease of the Abdominoperineal resection (APR)/Low anterior resection (LAR) ratio were observed in the second period. The overall pelvic recurrence rate was 25% in the first period and 11 in the second (P < 0.01). Significant differences were also found when the patients with LAR were compared between both periods, 30% vs 9% (P < 0.01) and specially when the 10 cm anal verge distance was considered to divide the LAR groups. No differences were found regarding the APR procedures in both periods. There was improved cancer-specific survival for the LAR group in the second period (P=0.03). CONCLUSION: Specialization and centralization influence the quality of rectal cancer surgery, mainly local recurrence rates and survival after low anterior resection.

Papel de la cirugía en el tratamiento del linfomagástrico primario / Surgery´s role in management of primary gastric lymphoma

El Linfoma Gástrico Primario es una entidad patológica cuyo manejo óptimo está en discusión. Se estudian retrospectivamente 23 pacientes tratados en nuestro Centro por Linfoma Gástrico entre 1976 y 1998 con resección quirúrgica como terapia principal. En diez pacientes la cirugía fue el único tratamiento realizado, en el resto se asoció quimio y/o radioterapia según criterio del oncólogo-hematólogo responsable. No hubo diferencias en cuanto a morbimortalidad entre los diferentes tratamientos. Se analizan las características clínicas e histológicas y la evolución de los pacientes. Ninguno de los pacientes ha fallecido a consecuencia del linfoma, no habiéndose presentado tampoco ningún caso de recurrencia local ni a distancia. Opinamos que la cirugía es una opción válida en el tratamiento del Linfoma Gástrico Primario. La asociación de tratamientos complementarios dependerá del estadio definitivo, las características histológicas del tumor y la posibilidad de realizar o no una resección radical (AU)

Serological prevalence to six leptospiral serovars in cattle in Asturias (Northern Spain).

The prevalence of antibody to six serovars of Leptospira interrogans in cattle in Asturias (Northern Spain) was determined by the microscopic agglutination test (MAT). Using 50% agglutination or lysis at a dilution of 1:10 or more as the criteria for seropositivity, 371 of 3578 (10.36%) animals were found to react with one serovar. The most commonly detected serovars were pomona (5.59%) and grippotyphosa (2.37%), whilst serovar hardjo (0.75%), icterohaemorrhagiae (0.64%), poi (0.64%) and autumnalis (0.36%) were found at lower frequencies.

Simple technique for the intraoperative detection of Crohn's strictures with a calibration sphere.

Several methods have been used to detect and evaluate small-bowel strictures in Crohn's disease. We describe a simple technique for the calibration of strictures using a 2.5-cm medical plastic sphere. This method provides an aseptic, safe, and effective calibration of the entire small bowel.

Immunohistochemical localisation of rabbit haemorrhagic disease virus VP-60 antigen in early infection of young and adult rabbits.

This study evaluated the time course distribution of rabbit haemorrhagic disease virus (RHDV) structural protein VP60 in tissues from experimentally infected rabbits from three different age groups. Viral VP60 antigen could not be detected in tissue samples from animals under four weeks, and only a few hepatocytes (0.01 to 0.2 per cent) were stained in the 6-week-old animals. A 6-week-old rabbit euthanised at 72 hpi showed VP60-labelling in hepatocytes and macrophages close to areas of inflammation. Viral VP60 antigen was detected as early as 12 hpi in a few hepatocytes (0.03 per cent) from adult animals. Within this age group, the extent of hepatocyte labelling considerably increased at 18 (3.0 per cent), 24 (25.5 per cent), 36 (50 per cent) and 48 (60 per cent) hpi. Extrahepatic viral VP60 antigen was also detected at 36 and 48 hpi in spleen macrophages and lymphocytes from adult rabbits. These findings support the hypothesis that the hepatocyte is the only cell type in the liver able to support RHDV replication almost immediately after viral infection.

[Role of surgery in the treatment of primary gastric lymphoma]. / Papel de la cirugía en el tratamiento del linfoma gástrico primario.

Primary gastric lymphoma's optimum management remains controversial. We reviewed our series of 23 patients with primary gastric lymphoma treated in our hospital between 1976 and 1998 with surgery as main therapy. Ten patients underwent surgical resection alone, whereas 13 also received postoperative adjuvant therapy, depending on the oncologist-haematologist's recommendations. No differences were found between treatments regarding mortality and morbidity. Clinical-histological features and patients, follow-up are analyzed. No patient died because of lymphoma and there wasn't either local or distant recurrence. We consider that surgery remains a valid option for the primary gastric lymphoma treatment. The introduction of combined modalities of radiation therapy and chemotherapy will depend on the final stage, the tumor histological features, and the feasibility of getting a radical resection.