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.

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.

[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.

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.

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.

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.

Effect of ivermectin treatment on anti-hypodermin C titers of Asturiana cattle naturally infected with Hypoderma lineatum.

The collagenase (hypodermin C) from soluble crude extracts of Hypoderma lineatum 1st-instar larvae was purified by reverse-phase HPLC and used in a new indirect ELISA test. This pure protein had several advantages over the use of crude larval extracts allowing a much better discrimination between infested and non-infested cattle. The anti-hypodermin C titers of 19 Asturiana cattle were estimated over the course of a natural H. lineatum infestation cycle, in which the effect of ivermectin treatment was also investigated. The results showed differences in the onset and ending of the infestation with respect to those described for other European countries. The ivermectin treatment proved to be very effective and treated animals had relatively low anticollagenase titers.

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.

Acute necrotizing pancreatitis: a multicenter study.

A multicenter study of acute necrotizing pancreatitis (ANP) classified in accordance with the Balthazar criteria (grades D and E), has been performed in 12 teaching hospitals. A total of 233 patients were reviewed, and the mortality rate was 26.6%. The most common etiology was biliary pancreatitis (45.5%). Among the complications, shock, renal insufficiency, pulmonary insufficiency and hemorrhagic gastritis were associated with a mortality rate of 51-66%. Diffuse fluid collections were associated with a higher mortality rate (26.8%) than localized fluid collections (14.5%). In 106 patients with gallstone pancreatitis, early surgery was performed in 17, and 5 patients (29.4%) died. No mortality was observed in 32 patients with delayed surgery. Sphincterotomy was performed in 13 patients, and 4 (30.7%) died. Early surgery (necrosectomy and closed peritoneal lavage) was undertaken in 75 patients, with a mortality rate of 39%. In conclusion, the morbidity and mortality rates of ANP can be improved with proper monitoring, adequate supportive care and the judicious use of surgery based on clinical and morphological findings.

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.

[Somatostatin prophylaxis following cephalic duodenopancreatectomy]. / Profilaxis con Somatostatina tras duodenopancreatectomía cefálica.

This study was aimed at assessing the value of prophylactic somatostatin after cephalic duodenopancreatectomy. A randomized prospective study was undertaken using two groups of patients, one with prophylactic somatostatin (4.5 mg daily in continuous perfusion for 7 days postoperatively), known as group I, and group II, which did not receive somatostatin. During a five-year period, from April 1989 to April 1994, we performed 35 duodenopancreatectomies, of which 21 belonged to group I and 14 to group II. We found a lower incidence of pancreatic anastomosis fistulae in group I (9.5% vs 35.7%; p < 0.05). We did not find any correlation between prophylaxis with somatostatin and the appearance of other complications or postoperative mortality. The mean time of fistula closure in group I was 5 days while that of group II was 19.2 days. In conclusion, the administration of prophylactic somatostatin after cephalic duodenopancreatectomy reduces the incidence and duration of pancreatic fistula.

[Breast cancer: value of various examinations in the diagnosis of the primary lesion]. / Cáncer de mama: valor de distintas exploraciones en el diagnóstico de la lesión primaria.

Breast physical examination which is enormous help in clinical diagnosis of cancer of the breast is not useful in the diagnosis of early lesions. We analized in this study different complementary examinations we do for the diagnosis of the cancer of the breast. Mammography, though maintaining a 5-7% false negative results is the examination of choice of the breast. Not with standing its simplicity, its contribution with respect to breast and neoplastic biology makes it a useful investigation. The diagnosis obtained through mammography in non palpable tumors permit the use of conservative therapeutic techniques at the same time allowing improvement in curation and survival rates when we deal preferently with Stage I cancer. It is still necessary in our means an information-sensibilization of the female population about breast autoexamination and the need for early specialized medical consultation. Any abnormal breast sign should alert us that more than 2.5 months delay would suppose a difference from a T1 to a T2 tumor.

[Follow-up of breast cancer: review of 750 cases after 5 years]. / Seguimiento del cáncer de mama: revisión de 750 casos a 5 años.

Recurrence of breast cancer can present in very small tumors even 20 years after initial treatment. Periodic revision of all the operated population during a long time will be necessary to detect all the recurrences. For this the cost-benefit relation of follow-up in breast cancer is a controversial topic. We present our results in 750 cases of breast cancer operated from 1980 and submitted to a follow-up protocol during five years. We analyzed the value of follow-up for the discovery of metastases, local recurrence after conservative treatment and after mastectomy. Finally we considered the usefulness of early diagnosis of familial breast cancer and cancer in the contralateral breast. Metastases was discovered in asymptomatic patients in 68%, which could improve the survival. Recurrence after mastectomy was seen in 1.3% of the patient and a half of these after treatment presented survival superior to 3 years. Follow-up favours early diagnosis and could have influence on survival.

[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.