The use of COLD-PCR, DHPLC and GeneScanning for the highly sensitive detection of c-KIT somatic mutations in canine mast cell tumours.

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different 'driver' somatic mutations of c-KIT, together with the wild-type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild-type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50-20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5-1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.

Prognostic significance of Kit receptor tyrosine kinase dysregulations in feline cutaneous mast cell tumors.

Feline cutaneous mast cell tumors (FeCMCTs) are characterized by variable biological behavior. Development of multiple nodules and potential visceral involvement, along with inconsistency of conventional prognostic aids, justify uncertainty in differentiating benign from malignant forms. c-Kit proto-oncogene activating mutations have been reported in feline mast cell tumors (MCTs), but their prognostic relevance was not investigated. This study was performed on FeCMCTs with variable clinical outcome to assess whether Kit cytoplasmic immunohistochemical labeling can be regarded as indicative of c-Kit mutations and to evaluate the relationship between Kit dysregulation and survival. Twenty-four cats diagnosed with a primary cutaneous MCT were enrolled. Kit immunohistochemical pattern and c-Kit (exons 8, 9, 11) mutational status were assessed in 34 tumor samples. Risk factors affecting survival were a number of mitoses greater than 5 per 10 HPFs (P = .017) and cytoplasmic Kit labeling (P = .045). Increased mitotic activity was associated with Kit cytoplasmic expression (P = .01). c-Kit encoding mutations were present in 19 (56%) tumors (exon 8, 19%; exon 9, 71%; exon 11, 10%), however, they were not significantly related to protein expression and they had no influence on prognosis. Additionally, in 6 of 9 (67%) cats, multiple nodules from the same cat had different mutational statuses. Mutations in the fifth immunoglobulin-like domain of Kit occur frequently in FeCMCT, but they are variably associated with aberrant protein expression and do not appear to be strictly correlated with biological behavior. These findings need to be confirmed in larger series, and exploration of further genomic regions of c-Kit is warranted.

Validation of a model to develop a symptom index for benign prostatic hyperplasia in dogs.

Benign prostatic hyperplasia (BPH) is a spontaneous and age-related condition in humans and intact male dogs. A symptom index for BPH in men was created by the American Urological Association. In this study, it has been developed and statistically validated as a model to assign an objective score to canine BPH severity based on clinical signs observed and/or subjectively reported to the veterinarian by dog owners. The medical records of the Animal Reproduction Unit of University of Bologna (Italy) were used to select dogs with a clinical diagnosis of BPH. A data set was built up, and the animals were included in the statistical analysis as dependent variables. A score of 1-3 was assigned to the disease severity of each case based on signs annotated, graded using a scale ranging from 1 to 4. Signs of BHP were entered as predictors while disease severity as dependent variable to generate the predictive model. The model was finally used to re-classify each case of the data set, and the percentage of corrected predictions calculated. Overall, 373 subjects were entered in the model. Between them, 243, 107 and 23 animals have been represented based on medical records with a BPH severity score of 1, 2 and 3, respectively. The model correctly predicted the response variable in 97.3% of the cases. In this study, a BPH symptom index was created for the first time in dogs, which may be useful to standardize BPH severity with an objective score and to evaluate the necessity, the kind and the effectiveness of treatment.

Cytological, immunohistochemical and mutational analysis of a gastric gastrointestinal stromal tumour in a cat.

Gastrointestinal stromal tumours (GISTs) represent a distinctive group of primary mesenchymal tumours of the gastrointestinal tract identified immunohistochemically by expression of CD117. A 10-year-old neutered female domestic shorthair cat with a history of recurrent vomiting was examined. The presence of a gastric mass was recognized and a laparotomy was performed. Cytological examination was consistent with a low-grade malignant mesenchymal tumour and histopathological investigation suggested myogenic differentiation of tumour cells. The diagnosis of GIST was confirmed by immunohistochemical expression of CD117. Sequence analysis of the KIT gene identified a deletion in exon 11. The same mutation is found often in human GISTs.

[Educational campaign targeting high school smokers]. / Intervento educativo rivolto a giovani fumatori della scuola secondaria di secondo grado.

The problem of smoking among young people continues to be a major health problem. In this study, high school students from the provinces of Rimini and Ravenna were invited to take the carbon monoxide test, in order to investigate the main reasons behind their smoking behaviour and to analyse attitudes towards changing the habit. The initiative involved 288 students: 56% males and 44% females aged between 14 and 20. The average daily consumption of cigarettes was 7.9 for the 14-17 age range, and 8.5 for the older group; the average age of starting smoking was 14.6 years. The consumption was seen to rise in both sexes at the weekend among those who smoked more than 10 cigarettes a day. The main reasons for smoking were relaxation and pleasure; the females were more addicted to cigarettes and to the rituals surrounding the habit. Although the students were aware of the risks involved in smoking, only 15% intended to quit. This confirms that awareness of the dangers alone is not sufficient to break the habit. Once again the school environment appears to be the most suitable place to meet young people in order to establish and assess their needs, and to set up programs of health promotion and education.

Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features.

Although the occurrence of thrombosis in acute promyelocytic leukemia (APL) has been reported during retinoic acid treatment, no studies carried out in large clinical cohorts have specifically addressed this issue. We analyzed 124 APL patients treated with the all-trans retinoic acid and idarubicin protocol and compared clinico-biologic characteristics of 11 patients who developed thrombosis with those of 113 patients who had no thrombosis. In seven patients, the events were recorded during induction, whereas in four patients deep vein thrombosis occurred in the post-induction phase. Comparison of clinico-biological characteristics of patients with and without thrombosis revealed in the former group higher median white blood cell (WBC) count (17 x 10(9)/l, range 1.2-56, P=0.002), prevalence of the bcr3 transcript type (72 vs 48%, P=0.01), of FLT3-ITD (64 vs 28%, P=0.02), CD2 (P=0.0001) and CD15 (P=0.01) expression. No correlation was found with sex, age, French-American-British subtype, all-trans-retinoic acid syndrome or with thrombophilic state that was investigated in 5/11 patients. Our findings suggest that, in APL patients consistent biologic features of leukemia cells may predict increased risk of developing thrombosis.

Discontinuation of imatinib therapy after achievement of complete molecular response in a Ph(+) CML patient treated while in long lasting complete cytogenetic remission (CCR) induced by interferon.

Imatinib has become the gold standard therapy for Ph(+) CML, as it induces complete cytogenetic remission (CCR) in 75-90% of patients in chronic phase (CP), and up to 40% of these patients obtain at least a 3 log reduction of BCR/ABL transcript [Kantarjian HM, Cortes JE, O'Brien S, Luthra R, Giles F, Verstovsek S, et al. Long-term survival benefit and improved complete cytogenetic and molecular response rates with imatinib mesylate in Philadelphia chromosome-positive chronic-phase chronic myeloid leukemia after failure of interferon-alpha. Blood. 2004;104:1979-1988]. However, it is not yet stated whether continued therapy is required to maintain this response or whether imatinib may be discontinued after confirmation of a prolonged complete molecular remission (CMR). We here report on a Ph(+) CML case in long lasting CCR following interferon-alpha treatment (IFN) which reached CMR with imatinib but soon relapsed at molecular level after this latter drug discontinuation; we considered the present observation also in the light of previously reported data.

Different biological behaviour of Waldenström Macroglobulinemia in two dogs.

Waldenström Macroglobulinemia is a low-grade immunosecretory disorder associated with lymphoid tumours, which is rarely reported in veterinary medicine. In this study, we describe two clinical cases of this rare syndrome in dogs, each characterized by a different onset and clinical course. In one case, a hyperacute onset and aggressive behaviour of the neoplasm was observed. Absolute serum viscosity (SV) was retrospectively evaluated in order to explain clinical findings. Rotational viscosimetry showed good precision in measuring SV. Both dogs had SV values higher than a control groups of healthy dogs although only one subject developed hyperviscosity symptoms and complications. At high paraprotein concentrations, a slight reduction of the M-component was associated with a marked decrease in SV. Thus, this work suggests that SV assessment is a relevant tool for managing monoclonal gammopathies, whose usefulness should be further confirmed in larger cohorts of dogs.

Comportamento de leitoas gestantes submetidas a dietas com baixo ou alto nível de fibra e mantidas em gaiolas ou em baias / Behaviour of pregnant gilts fed low or high fiber diets and maintained in crates or stalls

Avaliou-se o comportamento de leitoas gestantes que receberam dietas com baixo (BF= 4,5 por cento) ou alto (AF= 13 por cento) conteúdo de fibra bruta, alojadas em gaiolas individuais ou baias coletivas. Foram consideradas a postura e esteriotipias adotadas pelas fêmeas em três momentos: 10 e 30 minutos após o final do consumo da manhã (10AP e 30AP) e 10 minutos antes do arraçoamento da tarde (10AN). Para as fêmeas em baias, não houve associação das dietas com postura ou esteriotipias (P>0,05). Naquelas em gaiolas, a dieta AF esteve associada à menor freqüência da posição de pé e maior freqüência da posição deitada (P<0,05), aos 10AP. Aos 30AP, a dieta BF e o alojamento em gaiolas estavam associados com o aumento da postura sentada e diminuição na deitada (P<0,05). Aos 10AN, houve aumento na postura de pé e diminuição na deitada das fêmeas em gaiolas, independentemente da dieta (P>0,05). Nas fêmeas em gaiolas, a dieta AF esteve associada à redução de esteriotipias aos 10AP e 30AP. O alojamento em baias coletivas esteve associado à maior ausência de esteriotipias (P<0,05) aos 30AP. Aos 10AN, a dieta ou o local de alojamento não estavam associados com a ocorrência de esteriotipias (P>0,05). A dieta AF, quando administrada às fêmeas em gaiolas, reduziu a postura de pé aos 10AP e aumentou a ausência de esteriotipias aos 10AP e 30AP, o que sugere aumento do bem-estar das leitoas. A ansiedade das fêmeas em gaiolas, expressa pela maior ocorrência da postura de pé, aos 30AN, nào foi reduzida com a dieta AF.

Tobacco smoking among high school students in Romagna (Italy) and evaluation of a prevention campaign.

The aim of this study was to investigate the smoking habits of a sample of second year high school students in Romagna (Northern Italy) by means of an anonymous self-administered questionnaire and the efficiency of a previous health education campaign. A sample of 2,691 16-year-old pupils (74% of the school population of this age) was randomly selected. Of these, 863 (32.1%) had participated in a prevention campaign while attending middle school. 19.1% of the students who had taken part in the campaign were smokers compared to 23.2% of those who had not. The students' smoking habits were found to be influenced by the type of school and by the role models (parents, siblings, friends, teachers). The campaign appears to have had good results, especially in places where it was carried out on a wide scale. Suggestions are made for making such campaigns more efficient.

Smoking habits, exposure to passive smoking and attitudes to a non-smoking policy among hospital staff.

A survey was carried out into the smoking habits and exposure to passive smoking among health staff in the hospitals of Faenza, Forli and Rimini (Emilia-Romagna, Northern Italy), 2453 subjects answered anonymously a 41 question questionnaire. 53% of the subjects were professionals nurses, 16% doctors, 15% maintenance staff, 10% ancillary staff, 1% non-medical graduates, 2% were administrators and 3% were assigned to the category ¿other'. Of the subjects answering the questionnaire 39% were smokers, 19% ex-smokers and 42% non smokers. The highest number of smokers was found among women (41%) compared to men (37%) and among ancillary staff (48%) compared to nurses (41%) and doctors (31%). The males were mostly heavy smokers (> or = 20 cigarettes/d) and smoked strong cigarettes (> or = 12 mg/cig condensate content). The females were mostly light smokers (< 10 cigarettes/d) and smoked light cigarettes (1-6 mg/cig condensate content). A high percentage of subjects (87%) smoked at work especially in areas reserved for staff. 43% and 26% of shift workers and non-shift workers tended not to modify their habit when on morning or afternoon shifts. During night shifts the majority of them increased their tobacco consumption. Around 87% of hospital employees stated they were exposed to passive smoking inside the hospital especially in cooking areas, at information desks and corridors. Nurses, ancillaries and maintenance staff were those most exposed and for a greater number of hours per day compared to doctors. Almost all subjects were aware of the harm caused by passive smoking. 56% of smokers, 65% of ex-smokers and 72% of non smokers said they were willing to participate in future campaigns to limit smoking in their hospitals.

[A proposal for monitoring smoking prevention programs with the use of indicators]. / Una proposta per il monitoraggio di programmi di prevenzione dell'abitudine al fumo con l'uso di indicatori.

Health administrators are aware that the adoption of prevention programs of proven efficacy does not, by itself, guarantee good results as several factors can reduce effectiveness in a specific contest. Outcome evaluation and performance monitoring is thus growing, also with the aim of a more efficient resource use. On these premises, we describe a proposal for performance monitoring of smoking prevention programs, developed from literature and original data from the authors. The system is made of three components: a) five questions to assess smoking behavior (internal coherence, (Cronbach alpha = 0.731; k ranging from 0.39 to 0.76): b) two indicators (smoking prevalence and precentage of students reached): c) theorical and practical (number to sample according to population size) information for homogeneous and efficient cluster sampling. Performance is evaluated on account of three possible determinants of the results and explicit criteria are given to reach five possible decisions. The system was tested in five Health Districts of the Emilia Romagna Region with a population of 6370 8th class students and guided further planning and implementation. Further implementation and use of the system will give us information about the utility of this proposal, that could also be adopted as a model for the evaluation of other school prevention programs.