New Insights on Diagnostic Reproducibility of Biphasic Mesotheliomas: A Multi-Institutional Evaluation by the International Mesothelioma Panel From the MESOPATH Reference Center.

INTRODUCTION: The 2015 WHO classification of tumors categorized malignant mesothelioma into epithelioid, biphasic (BMM), and sarcomatoid (SMM) for prognostic relevance and treatment decisions. The survival of BMM is suspected to correlate with the amount of the sarcomatoid component. The criteria for a sarcomatoid component and the interobserver variability between pathologists for identifying this component are not well described. In ambiguous cases, a "transitional" (TMM) subtype has been proposed but was not accepted as a specific subtype in the 2015 WHO classification. The aims of this study were to evaluate the interobserver agreement in the diagnosis of BMM, to determine the nature and the significance of TMM subtype, and to relate the percentage of sarcomatoid component with survival. The value of staining for BRCA-1-associated protein (BAP1) and CDKN2A(p16) fluorescence in situ hybridization (FISH) were also assessed with respect to each of the tumoral components. METHODS: The study was conducted by the International Mesothelioma Panel supported by the French National Cancer Institute, the network of rare cancer (EURACAN) and in collaboration with the International Association for the Study of Lung Cancer (IASLC). The patient cases include a random group of 42 surgical biopsy samples diagnosed as BMM with evaluation of SMM component by the French Panel of MESOPATH experts was selected from the total series of 971 BMM cases collected from 1998 to 2016. Fourteen international pathologists with expertise in mesothelioma reviewed digitally scanned slides (hematoxylin and eosin - stained and pan-cytokeratin) without knowledge of prior diagnosis or outcome. Cases with at least 7 of 14 pathologists recognizing TMM features were selected as a TMM group. Demographic, clinical, histopathologic, treatment, and follow-up data were retrieved from the MESOBANK database. BAP1 (clone C-4) loss and CDKN2A(p16) homozygous deletion (HD) were assessed by immunohistochemistry (IHC) and FISH, respectively. Kappa statistics were applied for interobserver agreement and multivariate analysis with Cox regression adjusted for age and gender was performed for survival analysis. RESULTS: The 14 panelists recorded a total of 544 diagnoses. The interobserver correlation was moderate (weighted Kappa = 0.45). Of the cases originally classified as BMM by MESOPATH, the reviewers agreed in 71% of cases (385 of 544 opinions), with cases classified as pure epithelioid in 17% (93 of 544), and pure sarcomatoid in 12% (66 of 544 opinions). Diagnosis of BMM was made on morphology or IHC alone in 23% of the cases and with additional assessment of IHC in 77% (402 of 544). The median overall survival (OS) of the 42 BMM cases was 8 months. The OS for BMM was significantly different from SMM and epithelioid malignant mesothelioma (p < 0.0001). In BMM, a sarcomatoid component of less than 80% correlated with a better survival (p = 0.02). There was a significant difference in survival between BMM with TMM showing a median survival at 6 months compared to 12 months for those without TMM (p < 0.0001). BAP1 loss was observed in 50% (21 of 42) of the total cases and in both components in 26%. We also compared the TMM group to that of more aggressive patterns of epithelioid subtypes of mesothelioma (solid and pleomorphic of our large MESOPATH cohort). The curve of transitional type was persistently close to the OS curve of the sarcomatoid component. The group of sarcomatoid, transitional, and pleomorphic mesothelioma were very close to each other. We then considered the contribution of BAP1 immunostaining and loss of CDKN2A(p16) by FISH. BAP1 loss was observed in 50% (21 of 41) of the total cases and in both component in 27% of the cases (11 of 41). There was no significant difference in BAP1 loss between the TMM and non-TMM groups. HD CDKN2A(p16) was detected in 74% of the total cases with no significant difference between the TMM and non-TMM groups. In multivariate analysis, TMM morphology was an indicator of poor prognosis with a hazard ratio = 3.2; 95% confidence interval: 1.6 - 8.0; and p = 0.003 even when compared to the presence of HD CDKN2A(p16) on sarcomatoid component (hazard ratio = 4.5; 95% confidence interval: 1.2 - 16.3, p = 0.02). CONCLUSIONS: The interobserver concordance among the international mesothelioma and French mesothelioma panel suggests clinical utility for an updated definition of biphasic mesothelioma that allows better stratification of patients into risk groups for treatment decisions, systemic anticancer therapy, or selection for surgery or palliation. We also have shown the usefulness of FISH detection of CDKN2A(p16) HD compared to BAP1 loss on the spindle cell component for the separation in ambiguous cases between benign florid stromal reaction from true sarcomatoid component of biphasic mesothelioma. Taken together our results further validate the concept of transitional pattern as a poor prognostic indicator.

Salmonella spp. contamination in commercial layer hen farms using different types of samples and detection methods.

The performance of detection methods (culture methods and polymerase chain reaction assay) and plating media used in the same type of samples were determined as well as the specificity of PCR primers to detected Salmonella spp. contamination in layer hen farms. Also, the association of farm characteristics with Salmonella presence was evaluated. Environmental samples (feces, feed, drinking water, air, boot-swabs) and eggs were taken from 40 layer hen houses. Salmonella spp. was most detected in boot-swabs taken around the houses (30% and 35% by isolation and PCR, respectively) follow by fecal samples (15.2% and 13.6% by isolation and PCR, respectively). Eggs, drinking water, and air samples were negative for Salmonella detection. Salmonella Schwarzengrund and S. Enteritidis were the most isolated serotypes. For plating media, relative specificity was 1, and the relative sensitivity was greater for EF-18 agar than XLDT agar in feed and fecal samples. However, relative sensitivity was greater in XLDT agar than EF-18 agar for boot-swab samples. Agreement was between fair to good depending on the sample, and it was good between isolation and PCR (feces and boot-swabs), without agreement for feed samples. Salmonella spp. PCR was positive for all strains, while S. Typhimurium PCR was negative. Salmonella Enteritidis PCR used was not specific. Based in the multiple logistic regression analyses, categorization by counties was significant for Salmonella spp. presence (P-value = 0.010). This study shows the importance of considering different types of samples, plating media and detection methods during a Salmonella spp. monitoring study. In addition, it is important to incorporate the sampling of floors around the layer hen houses to learn if biosecurity measures should be strengthened to minimize the entry and spread of Salmonella in the houses. Also, the performance of some PCR methods and S. Enteritidis PCR should be improved, and biosecurity measures in hen farms must be reinforced in the region of more concentrated layer hen houses to reduce the probability of Salmonella spp. presence.

[The French mesothelioma network from 1998 to 2013]. / Mésothéliome: les dispositifs en place en France « le réseau mésothéliome ¼ 1998-2013.

Mesothelioma is a rare disease less than 0.3% of cancers in France, very aggressive and resistant to the majority of conventional therapies. Asbestos exposure is nearly the only recognized cause of mesothelioma in men observed in 80% of case. In 1990, the projections based on mortality predicted a raise of incidence in mesothelioma for the next three decades. Nowadays, the diagnosis of this cancer is based on pathology, but the histological presentation frequently heterogeneous, is responsible for numerous pitfalls and major problems of early detection toward effective therapy. Facing such a diagnostic, epidemiological and medico-legal context, a national and international multidisciplinary network has been progressively set up in order to answer to epidemiological survey, translational or academic research questions. Moreover, in response to the action of the French Cancer Program (action 23.1) a network of pathologists was organized for expert pathological second opinion using a standardized procedure of certification for mesothelioma diagnosis. We describe the network organization and show the results during this last 15years period of time from 1998-2013. These results show the major impact on patient's management, and confirm the interest of this second opinion to provide accuracy of epidemiological data, quality of medico-legal acknowledgement and accuracy of clinical diagnostic for the benefit of patients. We also show the impact of these collaborative efforts for creating a high quality clinicobiological, epidemiological and therapeutic data collection for improvement of the knowledge of this dramatic disease.

Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies.

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx(2EDL933), stx(2vha), stx(2vhb), stx(2g), stx(1EDL933), and stx(1d) were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

[Drug-induced pneumonitis due to sirolimus: an interaction with atorvastatin?]. / Pneumopathie médicamenteuse sous sirolimus: interaction avec l'atorvastatine?

INTRODUCTION: Sirolimus is an immunosupressant used in renal transplantation because of its lack of nephrotoxicity. We report four cases of pneumonitis due to sirolimus, possibly revealing an interaction with atorvastatin. CASE REPORT: Four patients (previously on long-term treatment with atorvastatin) presented with respiratory symptoms between 3 and 56 months after starting treatment with sirolimus following renal transplantation. Thoracic CT scans showed bilateral areas of peripheral alveolar consolidation. Bronchial lavage showed a lymphocytic alveolitis. Open-lung biopsy showed organizing pneumonia associated with diffuse alveolar damage and caseating granulomata. We attributed the pneumonitis to sirolimus on account of clinical and radiological resolution within 1 to 6 months of stopping treatment. We raise the possibility of an association between sirolimus and atorvastatin by competition for their hepatic degradation pathway via cytochrome P450 3A4. CONCLUSION: Sirolimus causes drug-induced pneumonitis that is predominantly an organizing pneumonia. Atorvastatin may encourage its development by competition with sirolimus in the liver.

Antibodies anti-Shiga toxin 2 B subunit from chicken egg yolk: isolation, purification and neutralization efficacy.

Shiga toxins (Stx1 and Stx2) are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The aim of this study was to evaluate the antibodies against Stx2 obtained from egg yolks of laying hens immunized with a recombinant Stx2B subunit. A high specific response in serum was observed 25 days after the first immunization and IgY antibodies were extracted from day 47th and purified from egg yolk. A concentration of 0.84 mg of total IgY/ml of egg yolk was obtained, of which 8% were antigen specific. The ability of anti-Stx2B IgY to recognize Stx2B and Stx2 either in solid-phase or in solution were evaluated and compared with anti-Stx2B rabbit antibodies by Western blotting and ELISA. The protective efficacy of IgY against Stx2 was determined by in vitro and in vivo experiments. The results show that IgY was able to recognize Stx2B and Stx2 in denatured conditions, attached to a solid-phase and free in solution. The anti-Stx2B IgY could effectively block the biological activity of Stx2 on Vero cells and protect mice from Stx2 challenge. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. IgY technology could be an useful tool for research, diagnosis and therapy of EHEC infection.

Hatching success and embryonic mortality on lesser rhea (Pterocnemia pennata) farms in northern Patagonia, Argentina.

1. The hatching success and embryonic mortality of 724 lesser rhea eggs from 12 farms in northern Patagonia, Argentina were monitored during the 2000 breeding season. 2. Average infertility was 24.5%, embryonic mortality was 33.5%, hatchability of fertile eggs was 66.5% and the neonatal mortality was 57.0%. There were large variations between different farms. 3. Bacterial contamination was high and egg hatchability was also affected by the occurrence of oedematous and malformed embryos. 4. No significant relationships were found between embryonic loss, duration of egg storage, initial egg weight or weight loss of hatched and failed eggs. 5. The low productivity on lesser rhea farms is caused not only by embryonic mortality but also by a high incidence of infertile eggs and neonatal mortality. 6. Bacterial infection may not have been the most important cause of incubation failure, indicating that nutritional deficiencies and inbreeding may play an important role in the productivity of these farms.

[Efficacy of rituximab in lymphomatoid granulomatosis]. / Efficacité du Rituximab au cours d'une granulomatose lymphomatoïde.

INTRODUCTION: Lymphomatoid Granulomatosis is a rare and serious disease, now considered to be a B-cell lymphoma, which is frequently associated with Epstein-Barr virus infection. There is no consensus on treatment, which is usually based on steroid therapy, either alone or combined with cyclophosphamide and combination chemotherapy. CASE REPORT: We report the case of an asymptomatic patient diagnosed after the incidental discovery of bilateral nodular opacities on their chest x-ray. Physical examination and bronchoscopy were normal. The diagnosis of Lymphomatoid Granulomatosis was made on the basis of surgical lung biopsy. Immunohistochemical studies confirmed the B phenotype of the lymphoma with the identification of atypical large CD 20 positive cells. In situ hybridisation confirmed the presence of EBV. In this case the course of the disease was slow. Treatment with anti CD 20 monoclonal antibodies (rituximab) led initially to a reduction in parenchymal abnormalities and mediastinal adenopathy. CONCLUSION: This treatment, recently used in Lymphomatoid Granulomatosis with pulmonary involvement, has shown promising results. Rituximab can be used in combination chemotherapy as standard treatment for aggressive B-cell lymphoma.