Impact of different methodologies on the detection of point mutations in routine air-dried fine needle aspiration (FNA) smears.

Currently the best method to select suspicious thyroid nodules for surgery is fine needle aspiration (FNA) cytology. However, FNA cytology has some inherent limitations, which can partly be overcome by molecular analysis. Therefore, molecular testing for somatic mutations has emerged as the most promising approach for molecular FNA diagnostics. The objective of this methodological study was to evaluate the feasibility of detecting BRAF, NRAS, HRAS, and KRAS mutations from routine air-dried thyroid FNA smears, and to find an optimal method for detecting these mutations in FNA samples. DNA was extracted from 110 routine air-dried FNA smears and the corresponding surgically obtained formalin-fixed paraffin-embedded tissues. The presence of BRAF, NRAS, HRAS, and KRAS mutations was assessed by real-time PCRs and high resolution melting analysis, and/or pyrosequencing in comparison to real-time PCRs using hybridization probes and fluorescence melting curve analysis. The high-resolution melting-PCRs revealed a significantly lower number of PCR failures and questionable results, and detected more mutations than the PCRs using hybridization probes. The number of PCR failures ranging from 14-16% by high-resolution melting-PCRs could be further reduced to 5-14% by adding pyrosequencing assays. Moreover, pyrosequencing increased the specificity of the assays, up to 98-100%, while the sensitivity ranged between 32-63%. In summary, the mutation detection, especially in air-dried FNA samples, improves when using PCR assays in combination with high resolution melting analysis. Additional improvement can be obtained by subsequent pyrosequencing in comparison to previously described real-time PCRs using hybridization probes and fluorescence melting curve analysis.

Context-dependent responses to novelty in Rainbow trout (Oncorhynchus mykiss), selected for high and low post-stress cortisol responsiveness.

Previous studies in a rainbow trout model, selectively bred for high (HR) and low (LR) post stress plasma cortisol levels, have yielded data that are indicative of contrasting stress coping styles. Fish from the HR line have been suggested to display a more diverse behavioral repertoire in challenging situations than the LR counterpart. The present study addressed whether such variation in behavioral flexibility traits was evident in different experimental settings using these selection lines. The fish were subjected to three sets of challenges (novel object test, resident-intruder test and confinement stressor test), all which were repeated a week later. Introducing a novel object evoked a divergent behavioral response in association with feeding: fish from the LR line displayed consistently suppressed feed intake while the HR fish remained unaffected. This observation was found to be repeatable along with attack latency and movement activity from the resident-intruder and confinement stressor tests. These results indicate that the behavioral responses in this animal model are context-dependent and shed new light on the expression of behavioral flexibility.

The prognostic significance of histological features in oral squamous cell carcinoma.

BACKGROUND: Different factors predict nodal metastasis, recurrence and survival in oral cancer. The aim was to assess the prognostic value of histological features related to the primary tumour. METHODS: A total of 144 patients surgically treated at Odense University Hospital for oral cancer between 1999 and 2004 were included in the study. Postoperative radiation therapy was given in case of close and involved margins or high TNM stages (UICC 1997). Median follow-up time was 38 months. All surgical resections were reviewed and 22 histological characteristics were assessed. RESULTS: The predominant sites were floor of mouth (FOM, 39%) and lateral tongue (35%). Fifty-nine per cent had UICC97 stage I-II disease. Five-year cause-specific survival was observed in 65%. Nodal involvement at diagnosis was observed in 36% which was significantly related to grade, neural and vascular invasion; surgical margins and increasing tumour depth. A cut-off value of 2 mm (4 mm for FOM) separated patients without and with nodal metastasis at the time of diagnosis. However, on multivariate analysis, neck disease was only associated with tumour depth and grade. Cox analysis of local recurrence in the oral cavity over time showed that tumour diameter and surgical margins were significant predictors while cause-specific survival was related to diameter, depth of invasion, surgical margins and extracapsular spread (ECS). CONCLUSIONS: Tumour depth and grade were strong prognostic factors for nodal metastasis, independently of other histological features. Tumour diameter and margins independently predict local recurrences in the oral cavity as well as cause-specific survival. Nodal involvement and ECS were associated with adverse prognosis.

Atlantic salmon (Salmo salar L.) parr fed genetically modified soybeans and maize: Histological, digestive, metabolic, and immunological investigations.

Physiological and health related responses to dietary inclusion of genetically modified (GM) full-fat soybean meal (Roundup Ready; GM-soy) and maize (MON810 Bt-maize; GM-maize), as well as non-parental, untransformed lines (nGM-soy and nGM-maize D2), were evaluated in farmed Atlantic salmon (Salmo salar L.) parr during the first 8 months of feeding. Significant effects of dietary GM presence were only found in intestinal Na+-dependent d-glucose uptake and SGLT1 protein level in the region pyloric caeca in which the highest values were found in the GM-soy, intermediate in the nGM-soy, and lowest in the standard FM fed groups. Data from this study confirm that GM soybeans (RRS) and maize (MON810) at inclusion levels of about 6% appear to be as safe as commercially available nGM soy and maize in diets for Atlantic salmon parr. Results from studies with higher inclusion levels and with non-modified, isogenic or near-isogenic parental lines as control groups are pending.

Histological, digestive, metabolic, hormonal and some immune factor responses in Atlantic salmon, Salmo salar L., fed genetically modified soybeans.

The paper reports the second and final part of an experiment aiming to study physiological and health-related effects of genetically modified (GM) soybean meal (SBM) type Roundup Ready soybean (RRS) in diets for post-smolt Atlantic salmon. For 3 months salmon were fed diets containing 172 g kg(-1) full-fat SBM from RRS (GM-soy) or an unmodified, non-isogenic line (nGM-soy), or a reference diet with fishmeal as the sole protein source (FM). Slight differences in anti-nutrient levels were observed between the GM and nGM-soy. Histological changes were observed only in the distal intestine of the soy-fed fish. The incidence of moderate inflammation was higher in the GM-soy group (9 of 10 sampled fish) compared with the nGM-soy group (7 of 10). However, no differences in the concomitant decreases in activities of digestive enzymes located in the brush border (leucine aminopeptidase and maltase) and apical cytoplasm (acid phosphatase) of enterocytes or in the number of major histocompatibility complex class II+ cells, lysozyme activity, or total IgM of the distal intestine were observed. GM compared with nGM-soy fed fish had higher head kidney lysozyme (11,856 vs. 10,456 units g(-1) tissue) and a tendency towards higher acid phosphatase (0.45 vs. 0.39 micromol h(-1) kg(-1) body mass in whole tissue) activities, respectively. Plasma insulin and thyroxin levels, and hepatic fructose-1,6-bisphosphatase and ethoxyresorufin-O-deethylase activities were not significantly affected. It is not possible, however, to conclude whether the differences in responses to GM-soy were due to the genetic modification or to differences in soy cultivars in the soy-containing diets. Results from studies using non-modified, parental line soybeans as the control group are necessary to evaluate whether genetic modification of soybeans in diets poses any risk to farmed Atlantic salmon.

Response to soy: T-cell-like reactivity in the intestine of Atlantic salmon, Salmo salar L.

T-cell-mediated hypersensitivity could be central in soybean meal (SBM)-induced intestinal changes in salmon. However, tools for immunohistochemical detection of T cells have been lacking in teleosts, including Atlantic salmon. Application of a specific histochemical protocol allowed demonstration of T-cell-like reactivities in formalin-fixed, paraffin-embedded tissues using an antibody reacting to a conserved region of human CD3epsilon (Dako A0452). Characteristic staining was observed in cells of the thymus as well as distal intestine, skin, gills and spleen. These cells were negative for immunoglobulin M (IgM). Intestinal intraepithelial leucocytes were CD3epsilon positive. During the SBM-induced enteropathy, the mixed inflammatory infiltrate in the lamina propria of the distal intestine included many lymphocytes with a T-cell-like reactivity. Real-time polymerase chain reaction revealed significantly increased expression of a complex polypeptide (CD3pp), CD4 and CD8beta (P < 0.05) in the distal intestine of SBM-fed fish compared to fish meal-fed reference fish. Increased reactivity for extracellular IgM in the lamina propria and a positive material between the epithelial cells at the tips of the folds was observed, possibly due to leakage of IgM through an abrogated epithelial barrier. In conclusion, a T-cell-like response appears to be involved in this example of a food-sensitive enteropathy.

Sentinel lymph node biopsy in oral cancer: validation of technique and clinical implications of added oblique planar lymphoscintigraphy and/or tomography.

PURPOSE: To validate lymphatic mapping combined with sentinel lymph node biopsy as a staging procedure, and to evaluate the possible clinical implications of added oblique lymphoscintigraphy and/or tomography and test the intra- and interobserver reproducibility of lymphoscintigraphy. MATERIAL AND METHODS: Forty patients (17 F and 23 M, aged 32-90) with 24 T1 and 16 T2 squamous cell carcinoma of the oral cavity. Planar lymphoscintigraphy, emission and transmission tomography were performed. Detection and excision of the sentinel nodes were guided by a gamma probe. The sentinel nodes were step-sectioning and stained with hematoxylin and eosin and cytokeratin (CK 1). Histology and follow-up were used as "gold standard". Tumor location, number of sentinel lymph nodes, metastasis, and recurrences were registered. Two observers evaluated the lymphoscintigraphic images to assess the inter-rater agreement. RESULTS: Eleven (28%) patients were upstaged. The sentinel lymph node identification rate was 97.5%. Sentinel lymph node biopsy significantly differentiated between patients with or without lymph node metastasis (P = 0.001). Lymphatic mapping revealed 124 hotspots and 144 hot lymph nodes were removed by sentinel lymph node biopsy. Three patients developed a lymph node recurrence close to the primary tumor site during follow-up. Added oblique lymphoscintigraphic images and/or tomography revealed extra hotspots in 15/40 (38%) patients. In 4/40 (10%), extra contralateral hotspots were detected. CONCLUSION: Sentinel lymph node biopsy upstaged 28% of the patients. Sentinel lymph nodes close to the primary tumor were difficult to find. Added oblique planar images and/or tomographic images revealed extra clinical relevant hotspots in 38% of patients. Reproducibility proved excellent.

Staging N0 oral cancer: lymphoscintigraphy and conventional imaging.

PURPOSE: To compare sentinel lymph node biopsy, magnetic resonance imaging (MRI), Doppler ultrasonography, and palpation as staging tools in patients with T1/T2 N0 cancer of the oral cavity. MATERIAL AND METHODS: Forty consecutive patients were enrolled (17 F and 23 M, aged 32-90 years), 24 T1 and 16 T2 cN0 squamous cell carcinoma of the oral cavity. Palpation was carried out by two observers prior to inclusion. MRI, gray-scale and Doppler ultrasonography were performed. Lymphoscintigraphies were done after peritumoral injections of 99mTc labelled rheniumsulphide nanocolloid, followed by sentinel lymph node biopsy guided by a gamma probe and Patent Blue. Palpation, Doppler ultrasonography, MRI, and sentinel lymph node biopsy were compared to a combination of histopathology and follow-up. Diagnostic testing was performed using the x2 test. RESULTS: Histopathological examination revealed metastatic spread to the neck in 14 of 40 patients. One patient had bilateral neck disease. Sentinel lymph node biopsy and ultrasonography were performed in 80 neck sides of 40 patients and MRI in 70 neck sides (5 patients were claustrophobic). SN revealed suspicious lymph nodes in 12 necks, ultrasonography in 23 necks, and MRI in 9 necks. The positive predictive value of sentinel lymph node biopsy was 100%, ultrasonography 57%, and MRI 56%. The respective negative predictive values were 96%, 96%, and 85%. The sensitivity of sentinel lymph node biopsy 80% was comparable to ultrasonography 87%, but the sensitivity of MRI 36% was low. The specificities were 100%, 85%, and 93%, respectively. By combined sentinel lymph node biopsy and ultrasonography the overall sensitivity could have been 100%. CONCLUSION: Sentinel lymph node biopsy improved staging of patients with small N0 oral cancers. Combined sentinel lymph node biopsy and Doppler ultrasonography may further improve staging. MRI and simple palpation results were poor.

An examination of the intestinal tract of Atlantic salmon, Salmo salar L., parr fed different varieties of soy and maize.

This study was conducted to investigate the long-term effects of feeding plant products from both traditional breeding and from biotechnology on intestinal somatic indices, histology and cell proliferation in first-feeding Atlantic salmon, Salmo salar L. (initial weight 0.21 +/- 0.02 g). A standard fishmeal diet (standard fishmeal) was formulated to contain fishmeal as the sole protein source and suprex maize as the main starch source. Six experimental diets were then developed: two in which some of the fishmeal was replaced with commercially available, genetically modified Roundup Ready full-fat soybean meal (GM-soy) or commercially available, non-GM full-fat soybean meal (nGM-soy) at a level of 12.5% of the total diet, and four diets in which the suprex maize was replaced with two lines of GM-maize (Dekalb 1; D1 and Pioneer 1; P1), both products of event MON810, and their half-sibling non-GM counterparts (Dekalb 2; D2 and Pioneer 2; P2), at a level of 12.1% of total diet. Each diet was fed to fish in triplicate tanks and the experiment lasted for 8 months, during which the fish reached a final weight of 101-116 g. There was no significant effect of diet on the intestinal indices, nor were histological changes observed in the pyloric caeca or mid intestine. In the distal intestine, one of nine sampled fish fed nGM-soy showed moderate changes, two of nine sampled fish fed GM-soy showed changes, one with moderate and one with severe changes, and two of nine fish fed nGM-maize D2 had moderate changes. Using a monoclonal antibody against proliferating cell nuclear antigen (PCNA), cell proliferative responses to the experimental diets were assessed. In fish fed both soy diets, a significantly higher (P < 0.05) cell proliferation response was observed in the distal intestine concomitant with an increased localization of PCNA positive cells along the whole distal intestinal folds. The PCNA response among the nGM-soy group was significantly higher compared with all the other diet groups. In contrast, for fish exposed to dietary maize (type D) compared with fish fed the standard fishmeal, the soy-diets (GM-soy and nGM-soy) and maize (type P), a significantly lower (P < 0.05) cell proliferation response was observed in the distal intestine. Results indicated that the GM plant products investigated in this study, at about 12% inclusion level, were as safe as commercially available non-GM products, at least in terms of their effect on indices and histological parameters of the Atlantic salmon intestinal tract.

Loss of heterozygosity at 9q33 and hypermethylation of the DBCCR1 gene in oral squamous cell carcinoma.

The DBCCR1 gene at chromosome 9q33 has been identified as a candidate tumour suppressor, which is frequently targeted by promoter hypermethylation in bladder cancer. Here, we studied the possible involvement of DBCCR1 in the development of oral squamous cell carcinoma. DNA from 34 tumours was examined for loss of heterozygosity (LOH) at three markers surrounding DBCCR1 and for hypermethylation of the DBCCR1 promoter, using methylation-specific PCR and methylation-specific melting-curve analysis. LOH was found in 10 of 31 cases (32%), and DBCCR1 hypermethylation was present in 15 of 34 cases (44%). Hypermethylation of DBCCR1 was also present in three of seven epithelial tissues adjacent to the tumours, including two hyperplastic and one histologically normal epithelia. Furthermore, of four oral leukoplakias with dysplasia, one showed LOH at 9q33 and two showed DBCCR1 hypermethylation. These data suggest that LOH at 9q33 and hypermethylation of the DBCCR1 promoter are frequent and possibly early events in oral malignant development.

Causes for improvement in nutritive value of broiler chicken diets with whole wheat instead of ground wheat.

1. Two experiments were conducted with broiler chicks to compare nutritive values of ground and whole wheat diets containing titanium oxide as indigestible marker. 2. In Experiment 1, a wheat and soy isolate based cold-pelleted diet, in which all the wheat was ground in a hammer mill to pass through a 3 mm sieve, was compared with a diet in which about half (375 g/kg) of the ground wheat was taken out of the diet and supplied as whole wheat mixed into the diet after pelleting. 3. In Experiment 2, a diet based on ground wheat and soy isolate which was pelleted after preconditioning (final temperature 81 degrees C) was compared with a diet in which 500 g of the ground wheat was replaced by whole wheat mixed into the diet before pelleting. 4. In Experiment 1, replacement of ground wheat with whole wheat increased apparent metabolisable energy (AME) content and starch digestibility measured at ileal as well as faecal level, while weight gain and feed conversion efficiency (FCE) were not affected. Jejunal chyme showed increased amylase activity and bile salt concentration. 5. In Experiment 2, replacement of ground wheat with whole wheat increased dry matter content as well as bile content of gizzard, and stimulated an increase in gizzard as well as pancreas weight. Jejunal amylase and bile concentrations were not significantly affected and neither were growth, FCE or AME. 6. Results from this experiment indicate that the improved feed value sometimes observed with whole wheat may be associated with modulation of digestive processes resulting in increased pancreas and liver secretions.

Effects of oat hulls and wood shavings on digestion in broilers and layers fed diets based on whole or ground wheat.

1. An experiment was conducted to study the effects of inclusion of oat hulls in diets based on whole or ground wheat for broilers. Effects of wood shavings on layers were investigated in a further experiment. 2. Inclusion of oat hulls in wheat-based broiler diets did not affect weight gain. Feed conversion efficiency (FCE), corrected for insoluble fibre contents, was improved by oat hull inclusion. 3. Gizzard size increased with inclusion of oat hulls, whole wheat, wood shavings and grit. 4. Starch digestibility was significantly increased by inclusion of oat hulls for broilers, and by wood shavings for layers. 5. Wood shavings and whole wheat did not affect bile acid concentration of gizzard contents. However, the total amount of bile acids in gizzard increased with access to wood shavings due to an increase in the weight of gizzard contents, indicating an increased gastroduodenal reflux. 6. Fibre concentration was considerably higher in the gizzard contents than in the feed. 7. Duodenal particle size decreased with access to grit. No effect of hulls or whole wheat inclusion was found, indicating that all particles are ground to a certain critical size before leaving the gizzard.

A rare case of orbital involvement in Riedel's thyroiditis.

We report a case of Riedel's thyroiditis in a 59-yr-old Caucasian female. She presented hypothyroidism and a stony hard, painful goiter. Due to fever, a high sedimentation rate and a high C-reactive protein (CRP), as well as high levels of anti-thyroid peroxidase antibodies (anti-TPOab), differential diagnostic considerations included acute and subacute thyroiditis as well as Hashimoto's thyroiditis and thyroid malignancy. At the same time the patient had clinical and radiological features of bilateral orbital pseudotumor with lacrimal gland involvement. During L-thyroxine therapy orbital symptoms and signs improved and thyroid size decreased. This case report serves as a reminder of differential diagnostic considerations in the etiology of goiter as well as ophthalmopathy. Although extremely rare, orbital sclerosing fibrosis can be seen in conjunction with Riedel's thyroiditis as part of multifocal fibrosclerosis.

Plasma cell gingivitis apparently related to the use of khat: report of a case.

Plasma cell gingivitis (PCG) is characterized by massive infiltration of plasma cells into the subepithelial tissue. It is a rare condition; the cause of which is still not fully understood. A case of PCG is reported in the mandibular gingiva probably caused by chewing khat. This report is the first, as far as we know, that relates PCG to the use of khat. The histological examination revealed infiltration of polyclonal plasma cells without signs of fungus, tuberculosis or malignancy. It is concluded that the changes were compatible with an allergic-like reaction. The patient, a 30-year-old immigrant from Somalia, revealed in a subsequent consultation that he regularly used khat. The leaves are placed in the buccal sulcus. The PCG disappeared within two weeks of the use of khat being discontinued. Dental surgeons (periodontists) in Europe and the New World will, due to increasing immigration from Africa and the Arabian Peninsula, meet more patients who regularly use khat. This means that PCG and other khat related intraoral changes will become more common in the future.

Esthesioneuroblastoma--what is the optimal treatment?

A retrospective review was conducted on 13 patients with esthesioneuroblastoma (ENB), treated at our institution from 1977 to 1997. According to the Kadish classification, one patient was in stage A, 5 patients were classified as stage B and 7 patients were in stage C. Five-year disease-specific survival was found to be 51%. Forty-six percent of the patients experienced relapse and despite intensive salvage therapy, median survival after recurrences was only 12 months. This indicates the need for good primary control in local as well as distant disease. The role of pre- versus postoperative radiotherapy to secure good local control is discussed and compared with the literature, and treatment guidelines are proposed. The tumours were graded according to the Hyams' classification and its importance as a prognostic factor is briefly discussed.

Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss).

Groups of fresh- and seawater-adapted Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) were fed diets with (SBM diet) or without (control diet) extracted soybean meal (30% of protein substituted with SBM) for 3 weeks. Average fish size per group ranged from 597 to 1763 g. One tank or net pen per species, dietary group and water salinity was used. In vitro nutrient transport (D-glucose, the L-amino acids aspartate, lysine, methionine, phenylalanine and proline, and the dipeptide glycyl-sarcosine) was measured using intact tissue (everted sleeve method) from the different postgastric intestinal regions. The dimensions of the different intestinal regions were also measured for each treatment group. Results indicate that SBM causes decreased carrier-mediated transport and increased permeability of distal intestinal epithelium for the nutrients, and the capacity of this region to absorb nutrient was diminished. Salinity may also affect the relative contribution of carrier-mediated and independent uptake to total nutrient absorption.

Recurrent pregnancy-related upper airway obstruction caused by intratracheal ectopic thyroid tissue.

An unusual case of recurrent pregnancy-related thyroid growth stimulation is reported. A 27-year-old euthyroid woman had pulmonary symptoms, thought to be asthma during her first pregnancy, that improved postpartum. Bronchodilatators had no effect and symptoms recurred from gestational week 22 during her second pregnancy. Her 58-mL multinodular goiter (by ultrasound) was not thought to be responsible for her upper airway symptoms. Therefore, fiber laryngoscopy and computed tomographic (CT) scan were performed and revealed a 20 x 15 x 10 mm intratracheal tumor. After tracheostomy and microlaryngoscopy, benign goitrous thyroid tissue was removed through a tracheal fissure during gestational week 35. Postoperatively the patient had stopped medication and was without any pulmonary symptoms. The child was delivered by cesarean section in gestational week 39. Apgar score was normal and the child has developed normally. We believe that this case illustrates the recurrent effect of pregnancy-related thyroid tissue stimulation by a combination of increasing human chorionic gonadotropin (hCG) stimulation and iodine deficiency in a borderline iodine-deficient region. This is the first report on symptomatic intratracheal ectopic thyroid tissue diagnosed during pregnancy.

Laryngeal cancer and human papillomavirus: HPV is absent in the majority of laryngeal carcinomas.

Thirty laryngeal carcinomas from patients without pre-existing laryngeal papillomatosis were examined by PCR for the presence of HPV DNA. The utmost care was taken during sectioning of the tissue blocks and DNA-extraction in order to avoid false positive results. Three pairs of consensus primers were used: MY9/MY11, GP5+/GP6+ and CPI/CPII. HPV was detected in 1/30 carcinomas. The HPV type present could not be determined, but it was not type 6, 11, 13, 16, 18, 30, 31, 33, 35 or 45. In other studies the reported frequency of HPV in laryngeal carcinomas, as estimated by PCR, varies between 3-85%. The reasons for this unacceptable variation in reported results are discussed. The present results indicate that HPV DNA does not have a major role in malignant tumours of the larynx in patients without pre-existing recurrent laryngeal papillomatosis.

Laryngeal dysplasia and the human papillomavirus.

The relationship between the human papillomavirus (HPV) and cancer of the upper aerodigestive trace is controversial. There is no doubt that at least some nasal, oral, pharyngeal and laryngeal carcinomas contain HPV DNA. However, the diversity in the reported incidence of HPV in these tumours represents a major problem. In the present study we have examined 30 laryngeal dysplastic lesions for HPV by the polymerase chain reaction, using three different pairs of consensus primers (GP5+/GP6+, MY09/MY11 and CPI/CPII). The single HPV-positive specimen was from a recurrent laryngeal papilloma. The HPV type present could not be determined, but it was not type 6, 11, 16, 18, 30, 31, 33 or 45. Restriction fragment length polymorphy analysis suggested a yet unknown HPV-type in this lesion. This investigation does not support the idea of HPV as an oncogenic cofactor in most laryngeal carcinomas as HPV was present in only 1/30 precancerous laryngeal lesions.